SRX8807546 GSM4685464 SRP273093 PRJNA647773 SRS7073607 GSM4685464 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685464 GSM4685464 GEO Accession GSM4685464 SRX8807547 GSM4685465 SRP273093 PRJNA647773 SRS7073608 GSM4685465 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685465 GSM4685465 GEO Accession GSM4685465 SRX8807548 GSM4685466 SRP273093 PRJNA647773 SRS7073609 GSM4685466 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685466 GSM4685466 GEO Accession GSM4685466 SRX8807549 GSM4685467 SRP273093 PRJNA647773 SRS7073610 GSM4685467 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685467 GSM4685467 GEO Accession GSM4685467 SRX8807550 GSM4685468 SRP273093 PRJNA647773 SRS7073611 GSM4685468 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685468 GSM4685468 GEO Accession GSM4685468 SRX8807551 GSM4685469 SRP273093 PRJNA647773 SRS7073612 GSM4685469 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685469 GSM4685469 GEO Accession GSM4685469 SRX8807552 GSM4685470 SRP273093 PRJNA647773 SRS7073613 GSM4685470 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685470 GSM4685470 GEO Accession GSM4685470 SRX8807553 GSM4685471 SRP273093 PRJNA647773 SRS7073614 GSM4685471 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685471 GSM4685471 GEO Accession GSM4685471 SRX8807554 GSM4685472 SRP273093 PRJNA647773 SRS7073615 GSM4685472 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685472 GSM4685472 GEO Accession GSM4685472 SRX8807555 GSM4685473 SRP273093 PRJNA647773 SRS7073616 GSM4685473 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685473 GSM4685473 GEO Accession GSM4685473 SRX8807556 GSM4685474 SRP273093 PRJNA647773 SRS7073617 GSM4685474 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685474 GSM4685474 GEO Accession GSM4685474 SRX8807557 GSM4685475 SRP273093 PRJNA647773 SRS7073619 GSM4685475 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685475 GSM4685475 GEO Accession GSM4685475 SRX8807558 GSM4685476 SRP273093 PRJNA647773 SRS7073618 GSM4685476 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685476 GSM4685476 GEO Accession GSM4685476 SRX8807559 GSM4685477 SRP273093 PRJNA647773 SRS7073620 GSM4685477 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685477 GSM4685477 GEO Accession GSM4685477 SRX8807560 GSM4685478 SRP273093 PRJNA647773 SRS7073621 GSM4685478 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685478 GSM4685478 GEO Accession GSM4685478 SRX8807561 GSM4685479 SRP273093 PRJNA647773 SRS7073622 GSM4685479 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685479 GSM4685479 GEO Accession GSM4685479 SRX8807562 GSM4685480 SRP273093 PRJNA647773 SRS7073623 GSM4685480 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685480 GSM4685480 GEO Accession GSM4685480 SRX8807563 GSM4685481 SRP273093 PRJNA647773 SRS7073624 GSM4685481 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685481 GSM4685481 GEO Accession GSM4685481 SRX8807564 GSM4685482 SRP273093 PRJNA647773 SRS7073625 GSM4685482 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685482 GSM4685482 GEO Accession GSM4685482 SRX8807565 GSM4685483 SRP273093 PRJNA647773 SRS7073626 GSM4685483 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685483 GSM4685483 GEO Accession GSM4685483 SRX8807566 GSM4685484 SRP273093 PRJNA647773 SRS7073627 GSM4685484 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685484 GSM4685484 GEO Accession GSM4685484 SRX8807567 GSM4685485 SRP273093 PRJNA647773 SRS7073628 GSM4685485 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685485 GSM4685485 GEO Accession GSM4685485 SRX8807568 GSM4685486 SRP273093 PRJNA647773 SRS7073629 GSM4685486 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685486 GSM4685486 GEO Accession GSM4685486 SRX8807569 GSM4685487 SRP273093 PRJNA647773 SRS7073630 GSM4685487 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685487 GSM4685487 GEO Accession GSM4685487 SRX8807570 GSM4685488 SRP273093 PRJNA647773 SRS7073631 GSM4685488 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685488 GSM4685488 GEO Accession GSM4685488 SRX8807571 GSM4685489 SRP273093 PRJNA647773 SRS7073632 GSM4685489 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685489 GSM4685489 GEO Accession GSM4685489 SRX8807572 GSM4685490 SRP273093 PRJNA647773 SRS7073633 GSM4685490 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685490 GSM4685490 GEO Accession GSM4685490 SRX8807573 GSM4685491 SRP273093 PRJNA647773 SRS7073634 GSM4685491 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685491 GSM4685491 GEO Accession GSM4685491 SRX8807574 GSM4685492 SRP273093 PRJNA647773 SRS7073635 GSM4685492 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685492 GSM4685492 GEO Accession GSM4685492 SRX8807575 GSM4685493 SRP273093 PRJNA647773 SRS7073636 GSM4685493 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685493 GSM4685493 GEO Accession GSM4685493 SRX8807576 GSM4685494 SRP273093 PRJNA647773 SRS7073637 GSM4685494 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685494 GSM4685494 GEO Accession GSM4685494 SRX8807577 GSM4685495 SRP273093 PRJNA647773 SRS7073638 GSM4685495 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685495 GSM4685495 GEO Accession GSM4685495 SRX8807578 GSM4685496 SRP273093 PRJNA647773 SRS7073639 GSM4685496 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685496 GSM4685496 GEO Accession GSM4685496 SRX8807579 GSM4685497 SRP273093 PRJNA647773 SRS7073640 GSM4685497 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685497 GSM4685497 GEO Accession GSM4685497 SRX8807580 GSM4685498 SRP273093 PRJNA647773 SRS7073641 GSM4685498 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685498 GSM4685498 GEO Accession GSM4685498 SRX8807581 GSM4685499 SRP273093 PRJNA647773 SRS7073642 GSM4685499 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685499 GSM4685499 GEO Accession GSM4685499 SRX8807582 GSM4685500 SRP273093 PRJNA647773 SRS7073643 GSM4685500 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685500 GSM4685500 GEO Accession GSM4685500 SRX8807583 GSM4685501 SRP273093 PRJNA647773 SRS7073644 GSM4685501 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685501 GSM4685501 GEO Accession GSM4685501 SRX8807584 GSM4685502 SRP273093 PRJNA647773 SRS7073645 GSM4685502 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685502 GSM4685502 GEO Accession GSM4685502 SRX8807585 GSM4685503 SRP273093 PRJNA647773 SRS7073646 GSM4685503 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685503 GSM4685503 GEO Accession GSM4685503 SRX8807586 GSM4685504 SRP273093 PRJNA647773 SRS7073647 GSM4685504 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685504 GSM4685504 GEO Accession GSM4685504 SRX8807587 GSM4685507 SRP273093 PRJNA647773 SRS7073648 GSM4685507 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685507 GSM4685507 GEO Accession GSM4685507 SRX8807588 GSM4685509 SRP273093 PRJNA647773 SRS7073649 GSM4685509 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685509 GSM4685509 GEO Accession GSM4685509 SRX8807589 GSM4685512 SRP273093 PRJNA647773 SRS7073650 GSM4685512 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685512 GSM4685512 GEO Accession GSM4685512 SRX8807590 GSM4685514 SRP273093 PRJNA647773 SRS7073651 GSM4685514 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685514 GSM4685514 GEO Accession GSM4685514 SRX8807591 GSM4685517 SRP273093 PRJNA647773 SRS7073652 GSM4685517 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685517 GSM4685517 GEO Accession GSM4685517 SRX8807592 GSM4685520 SRP273093 PRJNA647773 SRS7073653 GSM4685520 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685520 GSM4685520 GEO Accession GSM4685520 SRX8807593 GSM4685522 SRP273093 PRJNA647773 SRS7073654 GSM4685522 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685522 GSM4685522 GEO Accession GSM4685522 SRX8807594 GSM4685524 SRP273093 PRJNA647773 SRS7073655 GSM4685524 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685524 GSM4685524 GEO Accession GSM4685524 SRX8807595 GSM4685525 SRP273093 PRJNA647773 SRS7073656 GSM4685525 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685525 GSM4685525 GEO Accession GSM4685525 SRX8807596 GSM4685526 SRP273093 PRJNA647773 SRS7073657 GSM4685526 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685526 GSM4685526 GEO Accession GSM4685526 SRX8807597 GSM4685527 SRP273093 PRJNA647773 SRS7073658 GSM4685527 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685527 GSM4685527 GEO Accession GSM4685527 SRX8807598 GSM4685528 SRP273093 PRJNA647773 SRS7073659 GSM4685528 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685528 GSM4685528 GEO Accession GSM4685528 SRX8807599 GSM4685529 SRP273093 PRJNA647773 SRS7073660 GSM4685529 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685529 GSM4685529 GEO Accession GSM4685529 SRX8807600 GSM4685530 SRP273093 PRJNA647773 SRS7073661 GSM4685530 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685530 GSM4685530 GEO Accession GSM4685530 SRX8807601 GSM4685531 SRP273093 PRJNA647773 SRS7073662 GSM4685531 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685531 GSM4685531 GEO Accession GSM4685531 SRX8807602 GSM4685532 SRP273093 PRJNA647773 SRS7073663 GSM4685532 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685532 GSM4685532 GEO Accession GSM4685532 SRX8807603 GSM4685533 SRP273093 PRJNA647773 SRS7073664 GSM4685533 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685533 GSM4685533 GEO Accession GSM4685533 SRX8807604 GSM4685534 SRP273093 PRJNA647773 SRS7073665 GSM4685534 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685534 GSM4685534 GEO Accession GSM4685534 SRX8807605 GSM4685535 SRP273093 PRJNA647773 SRS7073666 GSM4685535 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685535 GSM4685535 GEO Accession GSM4685535 SRX8807606 GSM4685536 SRP273093 PRJNA647773 SRS7073667 GSM4685536 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685536 GSM4685536 GEO Accession GSM4685536 SRX8807607 GSM4685537 SRP273093 PRJNA647773 SRS7073668 GSM4685537 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685537 GSM4685537 GEO Accession GSM4685537 SRX8807608 GSM4685538 SRP273093 PRJNA647773 SRS7073669 GSM4685538 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685538 GSM4685538 GEO Accession GSM4685538 SRX8807609 GSM4685539 SRP273093 PRJNA647773 SRS7073670 GSM4685539 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685539 GSM4685539 GEO Accession GSM4685539 SRX8807610 GSM4685540 SRP273093 PRJNA647773 SRS7073672 GSM4685540 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685540 GSM4685540 GEO Accession GSM4685540 SRX8807611 GSM4685541 SRP273093 PRJNA647773 SRS7073671 GSM4685541 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685541 GSM4685541 GEO Accession GSM4685541 SRX8807612 GSM4685542 SRP273093 PRJNA647773 SRS7073673 GSM4685542 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685542 GSM4685542 GEO Accession GSM4685542 SRX8807613 GSM4685543 SRP273093 PRJNA647773 SRS7073675 GSM4685543 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685543 GSM4685543 GEO Accession GSM4685543 SRX8807614 GSM4685544 SRP273093 PRJNA647773 SRS7073674 GSM4685544 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685544 GSM4685544 GEO Accession GSM4685544 SRX8807615 GSM4685545 SRP273093 PRJNA647773 SRS7073676 GSM4685545 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685545 GSM4685545 GEO Accession GSM4685545 SRX8807616 GSM4685546 SRP273093 PRJNA647773 SRS7073678 GSM4685546 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685546 GSM4685546 GEO Accession GSM4685546 SRX8807617 GSM4685547 SRP273093 PRJNA647773 SRS7073677 GSM4685547 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685547 GSM4685547 GEO Accession GSM4685547 SRX8807618 GSM4685548 SRP273093 PRJNA647773 SRS7073679 GSM4685548 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685548 GSM4685548 GEO Accession GSM4685548 SRX8807619 GSM4685549 SRP273093 PRJNA647773 SRS7073680 GSM4685549 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685549 GSM4685549 GEO Accession GSM4685549 SRX8807620 GSM4685550 SRP273093 PRJNA647773 SRS7073681 GSM4685550 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685550 GSM4685550 GEO Accession GSM4685550 SRX8807621 GSM4685551 SRP273093 PRJNA647773 SRS7073682 GSM4685551 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685551 GSM4685551 GEO Accession GSM4685551 SRX8807622 GSM4685552 SRP273093 PRJNA647773 SRS7073683 GSM4685552 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685552 GSM4685552 GEO Accession GSM4685552 SRX8807623 GSM4685553 SRP273093 PRJNA647773 SRS7073684 GSM4685553 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685553 GSM4685553 GEO Accession GSM4685553 SRX8807624 GSM4685554 SRP273093 PRJNA647773 SRS7073685 GSM4685554 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685554 GSM4685554 GEO Accession GSM4685554 SRX8807625 GSM4685555 SRP273093 PRJNA647773 SRS7073686 GSM4685555 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685555 GSM4685555 GEO Accession GSM4685555 SRX8807626 GSM4685556 SRP273093 PRJNA647773 SRS7073687 GSM4685556 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685556 GSM4685556 GEO Accession GSM4685556 SRX8807627 GSM4685557 SRP273093 PRJNA647773 SRS7073688 GSM4685557 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685557 GSM4685557 GEO Accession GSM4685557 SRX8807628 GSM4685558 SRP273093 PRJNA647773 SRS7073689 GSM4685558 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685558 GSM4685558 GEO Accession GSM4685558 SRX8807629 GSM4685559 SRP273093 PRJNA647773 SRS7073691 GSM4685559 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685559 GSM4685559 GEO Accession GSM4685559 SRX8807630 GSM4685560 SRP273093 PRJNA647773 SRS7073690 GSM4685560 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685560 GSM4685560 GEO Accession GSM4685560 SRX8807631 GSM4685561 SRP273093 PRJNA647773 SRS7073692 GSM4685561 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685561 GSM4685561 GEO Accession GSM4685561 SRX8807632 GSM4685562 SRP273093 PRJNA647773 SRS7073693 GSM4685562 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685562 GSM4685562 GEO Accession GSM4685562 SRX8807633 GSM4685563 SRP273093 PRJNA647773 SRS7073694 GSM4685563 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685563 GSM4685563 GEO Accession GSM4685563 SRX8807634 GSM4685564 SRP273093 PRJNA647773 SRS7073695 GSM4685564 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685564 GSM4685564 GEO Accession GSM4685564 SRX8807635 GSM4685565 SRP273093 PRJNA647773 SRS7073696 GSM4685565 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685565 GSM4685565 GEO Accession GSM4685565 SRX8807636 GSM4685566 SRP273093 PRJNA647773 SRS7073697 GSM4685566 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685566 GSM4685566 GEO Accession GSM4685566 SRX8807637 GSM4685567 SRP273093 PRJNA647773 SRS7073698 GSM4685567 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685567 GSM4685567 GEO Accession GSM4685567 SRX8807638 GSM4685568 SRP273093 PRJNA647773 SRS7073699 GSM4685568 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685568 GSM4685568 GEO Accession GSM4685568 SRX8807639 GSM4685569 SRP273093 PRJNA647773 SRS7073701 GSM4685569 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685569 GSM4685569 GEO Accession GSM4685569 SRX8807640 GSM4685570 SRP273093 PRJNA647773 SRS7073700 GSM4685570 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685570 GSM4685570 GEO Accession GSM4685570 SRX8807641 GSM4685571 SRP273093 PRJNA647773 SRS7073702 GSM4685571 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685571 GSM4685571 GEO Accession GSM4685571 SRX8807642 GSM4685572 SRP273093 PRJNA647773 SRS7073704 GSM4685572 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685572 GSM4685572 GEO Accession GSM4685572 SRX8807643 GSM4685573 SRP273093 PRJNA647773 SRS7073703 GSM4685573 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685573 GSM4685573 GEO Accession GSM4685573 SRX8807644 GSM4685574 SRP273093 PRJNA647773 SRS7073705 GSM4685574 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685574 GSM4685574 GEO Accession GSM4685574 SRX8807645 GSM4685575 SRP273093 PRJNA647773 SRS7073706 GSM4685575 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685575 GSM4685575 GEO Accession GSM4685575 SRX8807646 GSM4685576 SRP273093 PRJNA647773 SRS7073707 GSM4685576 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685576 GSM4685576 GEO Accession GSM4685576 SRX8807647 GSM4685577 SRP273093 PRJNA647773 SRS7073708 GSM4685577 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685577 GSM4685577 GEO Accession GSM4685577 SRX8807648 GSM4685578 SRP273093 PRJNA647773 SRS7073709 GSM4685578 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685578 GSM4685578 GEO Accession GSM4685578 SRX8807649 GSM4685579 SRP273093 PRJNA647773 SRS7073710 GSM4685579 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685579 GSM4685579 GEO Accession GSM4685579 SRX8807650 GSM4685580 SRP273093 PRJNA647773 SRS7073711 GSM4685580 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685580 GSM4685580 GEO Accession GSM4685580 SRX8807651 GSM4685581 SRP273093 PRJNA647773 SRS7073712 GSM4685581 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685581 GSM4685581 GEO Accession GSM4685581 SRX8807652 GSM4685582 SRP273093 PRJNA647773 SRS7073713 GSM4685582 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685582 GSM4685582 GEO Accession GSM4685582 SRX8807653 GSM4685583 SRP273093 PRJNA647773 SRS7073714 GSM4685583 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685583 GSM4685583 GEO Accession GSM4685583 SRX8807654 GSM4685584 SRP273093 PRJNA647773 SRS7073715 GSM4685584 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685584 GSM4685584 GEO Accession GSM4685584 SRX8807655 GSM4685585 SRP273093 PRJNA647773 SRS7073716 GSM4685585 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685585 GSM4685585 GEO Accession GSM4685585 SRX8807656 GSM4685586 SRP273093 PRJNA647773 SRS7073717 GSM4685586 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685586 GSM4685586 GEO Accession GSM4685586 SRX8807657 GSM4685587 SRP273093 PRJNA647773 SRS7073718 GSM4685587 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685587 GSM4685587 GEO Accession GSM4685587 SRX8807658 GSM4685588 SRP273093 PRJNA647773 SRS7073719 GSM4685588 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685588 GSM4685588 GEO Accession GSM4685588 SRX8807659 GSM4685589 SRP273093 PRJNA647773 SRS7073720 GSM4685589 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685589 GSM4685589 GEO Accession GSM4685589 SRX8807660 GSM4685590 SRP273093 PRJNA647773 SRS7073721 GSM4685590 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685590 GSM4685590 GEO Accession GSM4685590 SRX8807661 GSM4685591 SRP273093 PRJNA647773 SRS7073722 GSM4685591 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685591 GSM4685591 GEO Accession GSM4685591 SRX8807662 GSM4685592 SRP273093 PRJNA647773 SRS7073723 GSM4685592 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685592 GSM4685592 GEO Accession GSM4685592 SRX8807663 GSM4685593 SRP273093 PRJNA647773 SRS7073724 GSM4685593 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685593 GSM4685593 GEO Accession GSM4685593 SRX8807664 GSM4685594 SRP273093 PRJNA647773 SRS7073725 GSM4685594 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685594 GSM4685594 GEO Accession GSM4685594 SRX8807665 GSM4685595 SRP273093 PRJNA647773 SRS7073726 GSM4685595 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685595 GSM4685595 GEO Accession GSM4685595 SRX8807666 GSM4685596 SRP273093 PRJNA647773 SRS7073727 GSM4685596 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685596 GSM4685596 GEO Accession GSM4685596 SRX8807667 GSM4685597 SRP273093 PRJNA647773 SRS7073728 GSM4685597 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685597 GSM4685597 GEO Accession GSM4685597 SRX8807668 GSM4685598 SRP273093 PRJNA647773 SRS7073729 GSM4685598 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685598 GSM4685598 GEO Accession GSM4685598 SRX8807669 GSM4685599 SRP273093 PRJNA647773 SRS7073730 GSM4685599 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685599 GSM4685599 GEO Accession GSM4685599 SRX8807670 GSM4685600 SRP273093 PRJNA647773 SRS7073731 GSM4685600 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685600 GSM4685600 GEO Accession GSM4685600 SRX8807671 GSM4685601 SRP273093 PRJNA647773 SRS7073732 GSM4685601 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685601 GSM4685601 GEO Accession GSM4685601 SRX8807672 GSM4685602 SRP273093 PRJNA647773 SRS7073733 GSM4685602 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685602 GSM4685602 GEO Accession GSM4685602 SRX8807673 GSM4685603 SRP273093 PRJNA647773 SRS7073734 GSM4685603 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685603 GSM4685603 GEO Accession GSM4685603 SRX8807674 GSM4685604 SRP273093 PRJNA647773 SRS7073735 GSM4685604 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685604 GSM4685604 GEO Accession GSM4685604 SRX8807675 GSM4685605 SRP273093 PRJNA647773 SRS7073736 GSM4685605 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685605 GSM4685605 GEO Accession GSM4685605 SRX8807676 GSM4685606 SRP273093 PRJNA647773 SRS7073737 GSM4685606 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685606 GSM4685606 GEO Accession GSM4685606 SRX8807677 GSM4685607 SRP273093 PRJNA647773 SRS7073738 GSM4685607 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685607 GSM4685607 GEO Accession GSM4685607 SRX8807678 GSM4685608 SRP273093 PRJNA647773 SRS7073739 GSM4685608 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685608 GSM4685608 GEO Accession GSM4685608 SRX8807679 GSM4685609 SRP273093 PRJNA647773 SRS7073740 GSM4685609 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685609 GSM4685609 GEO Accession GSM4685609 SRX8807680 GSM4685610 SRP273093 PRJNA647773 SRS7073741 GSM4685610 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685610 GSM4685610 GEO Accession GSM4685610 SRX8807681 GSM4685611 SRP273093 PRJNA647773 SRS7073742 GSM4685611 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685611 GSM4685611 GEO Accession GSM4685611 SRX8807682 GSM4685612 SRP273093 PRJNA647773 SRS7073743 GSM4685612 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685612 GSM4685612 GEO Accession GSM4685612 SRX8807683 GSM4685613 SRP273093 PRJNA647773 SRS7073744 GSM4685613 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685613 GSM4685613 GEO Accession GSM4685613 SRX8807684 GSM4685614 SRP273093 PRJNA647773 SRS7073745 GSM4685614 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685614 GSM4685614 GEO Accession GSM4685614 SRX8807685 GSM4685615 SRP273093 PRJNA647773 SRS7073746 GSM4685615 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685615 GSM4685615 GEO Accession GSM4685615 SRX8807686 GSM4685616 SRP273093 PRJNA647773 SRS7073747 GSM4685616 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685616 GSM4685616 GEO Accession GSM4685616 SRX8807687 GSM4685617 SRP273093 PRJNA647773 SRS7073748 GSM4685617 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685617 GSM4685617 GEO Accession GSM4685617 SRX8807688 GSM4685618 SRP273093 PRJNA647773 SRS7073749 GSM4685618 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685618 GSM4685618 GEO Accession GSM4685618 SRX8807689 GSM4685619 SRP273093 PRJNA647773 SRS7073750 GSM4685619 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685619 GSM4685619 GEO Accession GSM4685619 SRX8807690 GSM4685620 SRP273093 PRJNA647773 SRS7073751 GSM4685620 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685620 GSM4685620 GEO Accession GSM4685620 SRX8807691 GSM4685621 SRP273093 PRJNA647773 SRS7073752 GSM4685621 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685621 GSM4685621 GEO Accession GSM4685621 SRX8807692 GSM4685622 SRP273093 PRJNA647773 SRS7073753 GSM4685622 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685622 GSM4685622 GEO Accession GSM4685622 SRX8807693 GSM4685623 SRP273093 PRJNA647773 SRS7073754 GSM4685623 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685623 GSM4685623 GEO Accession GSM4685623 SRX8807694 GSM4685624 SRP273093 PRJNA647773 SRS7073755 GSM4685624 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685624 GSM4685624 GEO Accession GSM4685624 SRX8807695 GSM4685625 SRP273093 PRJNA647773 SRS7073756 GSM4685625 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685625 GSM4685625 GEO Accession GSM4685625 SRX8807696 GSM4685626 SRP273093 PRJNA647773 SRS7073757 GSM4685626 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685626 GSM4685626 GEO Accession GSM4685626 SRX8807697 GSM4685627 SRP273093 PRJNA647773 SRS7073758 GSM4685627 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685627 GSM4685627 GEO Accession GSM4685627 SRX8807698 GSM4685628 SRP273093 PRJNA647773 SRS7073759 GSM4685628 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685628 GSM4685628 GEO Accession GSM4685628 SRX8807699 GSM4685629 SRP273093 PRJNA647773 SRS7073760 GSM4685629 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685629 GSM4685629 GEO Accession GSM4685629 SRX8807700 GSM4685630 SRP273093 PRJNA647773 SRS7073761 GSM4685630 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685630 GSM4685630 GEO Accession GSM4685630 SRX8807701 GSM4685631 SRP273093 PRJNA647773 SRS7073762 GSM4685631 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685631 GSM4685631 GEO Accession GSM4685631 SRX8807702 GSM4685632 SRP273093 PRJNA647773 SRS7073763 GSM4685632 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685632 GSM4685632 GEO Accession GSM4685632 SRX8807703 GSM4685633 SRP273093 PRJNA647773 SRS7073764 GSM4685633 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685633 GSM4685633 GEO Accession GSM4685633 SRX8807704 GSM4685634 SRP273093 PRJNA647773 SRS7073765 GSM4685634 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685634 GSM4685634 GEO Accession GSM4685634 SRX8807705 GSM4685635 SRP273093 PRJNA647773 SRS7073766 GSM4685635 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685635 GSM4685635 GEO Accession GSM4685635 SRX8807706 GSM4685636 SRP273093 PRJNA647773 SRS7073767 GSM4685636 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685636 GSM4685636 GEO Accession GSM4685636 SRX8807707 GSM4685637 SRP273093 PRJNA647773 SRS7073768 GSM4685637 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685637 GSM4685637 GEO Accession GSM4685637 SRX8807708 GSM4685638 SRP273093 PRJNA647773 SRS7073769 GSM4685638 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685638 GSM4685638 GEO Accession GSM4685638 SRX8807709 GSM4685639 SRP273093 PRJNA647773 SRS7073770 GSM4685639 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685639 GSM4685639 GEO Accession GSM4685639 SRX8807710 GSM4685640 SRP273093 PRJNA647773 SRS7073771 GSM4685640 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685640 GSM4685640 GEO Accession GSM4685640 SRX8807711 GSM4685641 SRP273093 PRJNA647773 SRS7073772 GSM4685641 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685641 GSM4685641 GEO Accession GSM4685641 SRX8807712 GSM4685642 SRP273093 PRJNA647773 SRS7073773 GSM4685642 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685642 GSM4685642 GEO Accession GSM4685642 SRX8807713 GSM4685643 SRP273093 PRJNA647773 SRS7073774 GSM4685643 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685643 GSM4685643 GEO Accession GSM4685643 SRX8807714 GSM4685644 SRP273093 PRJNA647773 SRS7073775 GSM4685644 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685644 GSM4685644 GEO Accession GSM4685644 SRX8807715 GSM4685645 SRP273093 PRJNA647773 SRS7073776 GSM4685645 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685645 GSM4685645 GEO Accession GSM4685645 SRX8807716 GSM4685646 SRP273093 PRJNA647773 SRS7073777 GSM4685646 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685646 GSM4685646 GEO Accession GSM4685646 SRX8807717 GSM4685647 SRP273093 PRJNA647773 SRS7073778 GSM4685647 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685647 GSM4685647 GEO Accession GSM4685647 SRX8807718 GSM4685648 SRP273093 PRJNA647773 SRS7073779 GSM4685648 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685648 GSM4685648 GEO Accession GSM4685648 SRX8807719 GSM4685649 SRP273093 PRJNA647773 SRS7073780 GSM4685649 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685649 GSM4685649 GEO Accession GSM4685649 SRX8807720 GSM4685650 SRP273093 PRJNA647773 SRS7073781 GSM4685650 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685650 GSM4685650 GEO Accession GSM4685650 SRX8807721 GSM4685651 SRP273093 PRJNA647773 SRS7073782 GSM4685651 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685651 GSM4685651 GEO Accession GSM4685651 SRX8807722 GSM4685652 SRP273093 PRJNA647773 SRS7073783 GSM4685652 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685652 GSM4685652 GEO Accession GSM4685652 SRX8807723 GSM4685653 SRP273093 PRJNA647773 SRS7073784 GSM4685653 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685653 GSM4685653 GEO Accession GSM4685653 SRX8807724 GSM4685654 SRP273093 PRJNA647773 SRS7073785 GSM4685654 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685654 GSM4685654 GEO Accession GSM4685654 SRX8807725 GSM4685655 SRP273093 PRJNA647773 SRS7073786 GSM4685655 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685655 GSM4685655 GEO Accession GSM4685655 SRX8807726 GSM4685656 SRP273093 PRJNA647773 SRS7073787 GSM4685656 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685656 GSM4685656 GEO Accession GSM4685656 SRX8807727 GSM4685657 SRP273093 PRJNA647773 SRS7073788 GSM4685657 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685657 GSM4685657 GEO Accession GSM4685657 SRX8807728 GSM4685658 SRP273093 PRJNA647773 SRS7073789 GSM4685658 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685658 GSM4685658 GEO Accession GSM4685658 SRX8807729 GSM4685659 SRP273093 PRJNA647773 SRS7073790 GSM4685659 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685659 GSM4685659 GEO Accession GSM4685659 SRX8807730 GSM4685660 SRP273093 PRJNA647773 SRS7073791 GSM4685660 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685660 GSM4685660 GEO Accession GSM4685660 SRX8807731 GSM4685661 SRP273093 PRJNA647773 SRS7073792 GSM4685661 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685661 GSM4685661 GEO Accession GSM4685661 SRX8807732 GSM4685662 SRP273093 PRJNA647773 SRS7073793 GSM4685662 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685662 GSM4685662 GEO Accession GSM4685662 SRX8807733 GSM4685663 SRP273093 PRJNA647773 SRS7073794 GSM4685663 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685663 GSM4685663 GEO Accession GSM4685663 SRX8807734 GSM4685664 SRP273093 PRJNA647773 SRS7073795 GSM4685664 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685664 GSM4685664 GEO Accession GSM4685664 SRX8807735 GSM4685665 SRP273093 PRJNA647773 SRS7073796 GSM4685665 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685665 GSM4685665 GEO Accession GSM4685665 SRX8807736 GSM4685666 SRP273093 PRJNA647773 SRS7073797 GSM4685666 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685666 GSM4685666 GEO Accession GSM4685666 SRX8807737 GSM4685667 SRP273093 PRJNA647773 SRS7073798 GSM4685667 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685667 GSM4685667 GEO Accession GSM4685667 SRX8807738 GSM4685668 SRP273093 PRJNA647773 SRS7073799 GSM4685668 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685668 GSM4685668 GEO Accession GSM4685668 SRX8807739 GSM4685669 SRP273093 PRJNA647773 SRS7073800 GSM4685669 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685669 GSM4685669 GEO Accession GSM4685669 SRX8807740 GSM4685670 SRP273093 PRJNA647773 SRS7073801 GSM4685670 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685670 GSM4685670 GEO Accession GSM4685670 SRX8807741 GSM4685671 SRP273093 PRJNA647773 SRS7073802 GSM4685671 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685671 GSM4685671 GEO Accession GSM4685671 SRX8807742 GSM4685672 SRP273093 PRJNA647773 SRS7073803 GSM4685672 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685672 GSM4685672 GEO Accession GSM4685672 SRX8807743 GSM4685673 SRP273093 PRJNA647773 SRS7073804 GSM4685673 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685673 GSM4685673 GEO Accession GSM4685673 SRX8807744 GSM4685674 SRP273093 PRJNA647773 SRS7073805 GSM4685674 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685674 GSM4685674 GEO Accession GSM4685674 SRX8807745 GSM4685675 SRP273093 PRJNA647773 SRS7073806 GSM4685675 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685675 GSM4685675 GEO Accession GSM4685675 SRX8807746 GSM4685676 SRP273093 PRJNA647773 SRS7073807 GSM4685676 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685676 GSM4685676 GEO Accession GSM4685676 SRX8807747 GSM4685677 SRP273093 PRJNA647773 SRS7073808 GSM4685677 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685677 GSM4685677 GEO Accession GSM4685677 SRX8807748 GSM4685678 SRP273093 PRJNA647773 SRS7073809 GSM4685678 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685678 GSM4685678 GEO Accession GSM4685678 SRX8807749 GSM4685679 SRP273093 PRJNA647773 SRS7073810 GSM4685679 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685679 GSM4685679 GEO Accession GSM4685679 SRX8807750 GSM4685680 SRP273093 PRJNA647773 SRS7073811 GSM4685680 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685680 GSM4685680 GEO Accession GSM4685680 SRX8807751 GSM4685681 SRP273093 PRJNA647773 SRS7073813 GSM4685681 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685681 GSM4685681 GEO Accession GSM4685681 SRX8807752 GSM4685682 SRP273093 PRJNA647773 SRS7073812 GSM4685682 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685682 GSM4685682 GEO Accession GSM4685682 SRX8807753 GSM4685683 SRP273093 PRJNA647773 SRS7073814 GSM4685683 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685683 GSM4685683 GEO Accession GSM4685683 SRX8807754 GSM4685684 SRP273093 PRJNA647773 SRS7073815 GSM4685684 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685684 GSM4685684 GEO Accession GSM4685684 SRX8807755 GSM4685685 SRP273093 PRJNA647773 SRS7073816 GSM4685685 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685685 GSM4685685 GEO Accession GSM4685685 SRX8807756 GSM4685686 SRP273093 PRJNA647773 SRS7073817 GSM4685686 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685686 GSM4685686 GEO Accession GSM4685686 SRX8807757 GSM4685687 SRP273093 PRJNA647773 SRS7073818 GSM4685687 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685687 GSM4685687 GEO Accession GSM4685687 SRX8807758 GSM4685688 SRP273093 PRJNA647773 SRS7073819 GSM4685688 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685688 GSM4685688 GEO Accession GSM4685688 SRX8807759 GSM4685689 SRP273093 PRJNA647773 SRS7073820 GSM4685689 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685689 GSM4685689 GEO Accession GSM4685689 SRX8807760 GSM4685690 SRP273093 PRJNA647773 SRS7073821 GSM4685690 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685690 GSM4685690 GEO Accession GSM4685690 SRX8807761 GSM4685691 SRP273093 PRJNA647773 SRS7073822 GSM4685691 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685691 GSM4685691 GEO Accession GSM4685691 SRX8807762 GSM4685692 SRP273093 PRJNA647773 SRS7073823 GSM4685692 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685692 GSM4685692 GEO Accession GSM4685692 SRX8807763 GSM4685693 SRP273093 PRJNA647773 SRS7073824 GSM4685693 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685693 GSM4685693 GEO Accession GSM4685693 SRX8807764 GSM4685694 SRP273093 PRJNA647773 SRS7073825 GSM4685694 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685694 GSM4685694 GEO Accession GSM4685694 SRX8807765 GSM4685695 SRP273093 PRJNA647773 SRS7073826 GSM4685695 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685695 GSM4685695 GEO Accession GSM4685695 SRX8807766 GSM4685696 SRP273093 PRJNA647773 SRS7073827 GSM4685696 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685696 GSM4685696 GEO Accession GSM4685696 SRX8807767 GSM4685697 SRP273093 PRJNA647773 SRS7073828 GSM4685697 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685697 GSM4685697 GEO Accession GSM4685697 SRX8807768 GSM4685698 SRP273093 PRJNA647773 SRS7073829 GSM4685698 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685698 GSM4685698 GEO Accession GSM4685698 SRX8807769 GSM4685699 SRP273093 PRJNA647773 SRS7073831 GSM4685699 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685699 GSM4685699 GEO Accession GSM4685699 SRX8807770 GSM4685700 SRP273093 PRJNA647773 SRS7073830 GSM4685700 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685700 GSM4685700 GEO Accession GSM4685700 SRX8807771 GSM4685701 SRP273093 PRJNA647773 SRS7073832 GSM4685701 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685701 GSM4685701 GEO Accession GSM4685701 SRX8807772 GSM4685702 SRP273093 PRJNA647773 SRS7073833 GSM4685702 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685702 GSM4685702 GEO Accession GSM4685702 SRX8807773 GSM4685703 SRP273093 PRJNA647773 SRS7073834 GSM4685703 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685703 GSM4685703 GEO Accession GSM4685703 SRX8807774 GSM4685704 SRP273093 PRJNA647773 SRS7073835 GSM4685704 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685704 GSM4685704 GEO Accession GSM4685704 SRX8807775 GSM4685705 SRP273093 PRJNA647773 SRS7073836 GSM4685705 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685705 GSM4685705 GEO Accession GSM4685705 SRX8807776 GSM4685706 SRP273093 PRJNA647773 SRS7073837 GSM4685706 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685706 GSM4685706 GEO Accession GSM4685706 SRX8807777 GSM4685707 SRP273093 PRJNA647773 SRS7073840 GSM4685707 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685707 GSM4685707 GEO Accession GSM4685707 SRX8807778 GSM4685708 SRP273093 PRJNA647773 SRS7073838 GSM4685708 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685708 GSM4685708 GEO Accession GSM4685708 SRX8807779 GSM4685709 SRP273093 PRJNA647773 SRS7073839 GSM4685709 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685709 GSM4685709 GEO Accession GSM4685709 SRX8807780 GSM4685710 SRP273093 PRJNA647773 SRS7073842 GSM4685710 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685710 GSM4685710 GEO Accession GSM4685710 SRX8807781 GSM4685711 SRP273093 PRJNA647773 SRS7073843 GSM4685711 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685711 GSM4685711 GEO Accession GSM4685711 SRX8807782 GSM4685712 SRP273093 PRJNA647773 SRS7073841 GSM4685712 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685712 GSM4685712 GEO Accession GSM4685712 SRX8807783 GSM4685713 SRP273093 PRJNA647773 SRS7073844 GSM4685713 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685713 GSM4685713 GEO Accession GSM4685713 SRX8807784 GSM4685714 SRP273093 PRJNA647773 SRS7073845 GSM4685714 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685714 GSM4685714 GEO Accession GSM4685714 SRX8807785 GSM4685715 SRP273093 PRJNA647773 SRS7073846 GSM4685715 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685715 GSM4685715 GEO Accession GSM4685715 SRX8807786 GSM4685716 SRP273093 PRJNA647773 SRS7073848 GSM4685716 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685716 GSM4685716 GEO Accession GSM4685716 SRX8807787 GSM4685717 SRP273093 PRJNA647773 SRS7073847 GSM4685717 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685717 GSM4685717 GEO Accession GSM4685717 SRX8807788 GSM4685718 SRP273093 PRJNA647773 SRS7073849 GSM4685718 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685718 GSM4685718 GEO Accession GSM4685718 SRX8807789 GSM4685719 SRP273093 PRJNA647773 SRS7073850 GSM4685719 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685719 GSM4685719 GEO Accession GSM4685719 SRX8807790 GSM4685720 SRP273093 PRJNA647773 SRS7073851 GSM4685720 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685720 GSM4685720 GEO Accession GSM4685720 SRX8807791 GSM4685721 SRP273093 PRJNA647773 SRS7073852 GSM4685721 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685721 GSM4685721 GEO Accession GSM4685721 SRX8807792 GSM4685722 SRP273093 PRJNA647773 SRS7073853 GSM4685722 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685722 GSM4685722 GEO Accession GSM4685722 SRX8807793 GSM4685723 SRP273093 PRJNA647773 SRS7073854 GSM4685723 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685723 GSM4685723 GEO Accession GSM4685723 SRX8807794 GSM4685724 SRP273093 PRJNA647773 SRS7073855 GSM4685724 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685724 GSM4685724 GEO Accession GSM4685724 SRX8807795 GSM4685725 SRP273093 PRJNA647773 SRS7073856 GSM4685725 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685725 GSM4685725 GEO Accession GSM4685725 SRX8807796 GSM4685726 SRP273093 PRJNA647773 SRS7073857 GSM4685726 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685726 GSM4685726 GEO Accession GSM4685726 SRX8807797 GSM4685727 SRP273093 PRJNA647773 SRS7073858 GSM4685727 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685727 GSM4685727 GEO Accession GSM4685727 SRX8807798 GSM4685728 SRP273093 PRJNA647773 SRS7073859 GSM4685728 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685728 GSM4685728 GEO Accession GSM4685728 SRX8807799 GSM4685729 SRP273093 PRJNA647773 SRS7073860 GSM4685729 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685729 GSM4685729 GEO Accession GSM4685729 SRX8807800 GSM4685730 SRP273093 PRJNA647773 SRS7073861 GSM4685730 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685730 GSM4685730 GEO Accession GSM4685730 SRX8807801 GSM4685731 SRP273093 PRJNA647773 SRS7073864 GSM4685731 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685731 GSM4685731 GEO Accession GSM4685731 SRX8807802 GSM4685732 SRP273093 PRJNA647773 SRS7073862 GSM4685732 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685732 GSM4685732 GEO Accession GSM4685732 SRX8807803 GSM4685733 SRP273093 PRJNA647773 SRS7073863 GSM4685733 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685733 GSM4685733 GEO Accession GSM4685733 SRX8807804 GSM4685734 SRP273093 PRJNA647773 SRS7073865 GSM4685734 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685734 GSM4685734 GEO Accession GSM4685734 SRX8807805 GSM4685735 SRP273093 PRJNA647773 SRS7073866 GSM4685735 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685735 GSM4685735 GEO Accession GSM4685735 SRX8807806 GSM4685736 SRP273093 PRJNA647773 SRS7073867 GSM4685736 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685736 GSM4685736 GEO Accession GSM4685736 SRX8807807 GSM4685737 SRP273093 PRJNA647773 SRS7073868 GSM4685737 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685737 GSM4685737 GEO Accession GSM4685737 SRX8807808 GSM4685738 SRP273093 PRJNA647773 SRS7073869 GSM4685738 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685738 GSM4685738 GEO Accession GSM4685738 SRX8807809 GSM4685739 SRP273093 PRJNA647773 SRS7073870 GSM4685739 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685739 GSM4685739 GEO Accession GSM4685739 SRX8807810 GSM4685740 SRP273093 PRJNA647773 SRS7073871 GSM4685740 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685740 GSM4685740 GEO Accession GSM4685740 SRX8807811 GSM4685741 SRP273093 PRJNA647773 SRS7073872 GSM4685741 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685741 GSM4685741 GEO Accession GSM4685741 SRX8807812 GSM4685742 SRP273093 PRJNA647773 SRS7073873 GSM4685742 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685742 GSM4685742 GEO Accession GSM4685742 SRX8807813 GSM4685743 SRP273093 PRJNA647773 SRS7073875 GSM4685743 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685743 GSM4685743 GEO Accession GSM4685743 SRX8807814 GSM4685744 SRP273093 PRJNA647773 SRS7073874 GSM4685744 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685744 GSM4685744 GEO Accession GSM4685744 SRX8807815 GSM4685745 SRP273093 PRJNA647773 SRS7073876 GSM4685745 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685745 GSM4685745 GEO Accession GSM4685745 SRX8807816 GSM4685746 SRP273093 PRJNA647773 SRS7073877 GSM4685746 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685746 GSM4685746 GEO Accession GSM4685746 SRX8807817 GSM4685747 SRP273093 PRJNA647773 SRS7073878 GSM4685747 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685747 GSM4685747 GEO Accession GSM4685747 SRX8807818 GSM4685748 SRP273093 PRJNA647773 SRS7073879 GSM4685748 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685748 GSM4685748 GEO Accession GSM4685748 SRX8807819 GSM4685749 SRP273093 PRJNA647773 SRS7073880 GSM4685749 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685749 GSM4685749 GEO Accession GSM4685749 SRX8807820 GSM4685750 SRP273093 PRJNA647773 SRS7073881 GSM4685750 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685750 GSM4685750 GEO Accession GSM4685750 SRX8807821 GSM4685751 SRP273093 PRJNA647773 SRS7073882 GSM4685751 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685751 GSM4685751 GEO Accession GSM4685751 SRX8807822 GSM4685752 SRP273093 PRJNA647773 SRS7073883 GSM4685752 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685752 GSM4685752 GEO Accession GSM4685752 SRX8807823 GSM4685753 SRP273093 PRJNA647773 SRS7073884 GSM4685753 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685753 GSM4685753 GEO Accession GSM4685753 SRX8807824 GSM4685754 SRP273093 PRJNA647773 SRS7073885 GSM4685754 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685754 GSM4685754 GEO Accession GSM4685754 SRX8807825 GSM4685755 SRP273093 PRJNA647773 SRS7073886 GSM4685755 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685755 GSM4685755 GEO Accession GSM4685755 SRX8807826 GSM4685756 SRP273093 PRJNA647773 SRS7073888 GSM4685756 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685756 GSM4685756 GEO Accession GSM4685756 SRX8807827 GSM4685757 SRP273093 PRJNA647773 SRS7073887 GSM4685757 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685757 GSM4685757 GEO Accession GSM4685757 SRX8807828 GSM4685758 SRP273093 PRJNA647773 SRS7073889 GSM4685758 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685758 GSM4685758 GEO Accession GSM4685758 SRX8807829 GSM4685759 SRP273093 PRJNA647773 SRS7073890 GSM4685759 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685759 GSM4685759 GEO Accession GSM4685759 SRX8807830 GSM4685760 SRP273093 PRJNA647773 SRS7073891 GSM4685760 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685760 GSM4685760 GEO Accession GSM4685760 SRX8807831 GSM4685761 SRP273093 PRJNA647773 SRS7073892 GSM4685761 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685761 GSM4685761 GEO Accession GSM4685761 SRX8807832 GSM4685762 SRP273093 PRJNA647773 SRS7073893 GSM4685762 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685762 GSM4685762 GEO Accession GSM4685762 SRX8807833 GSM4685763 SRP273093 PRJNA647773 SRS7073894 GSM4685763 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685763 GSM4685763 GEO Accession GSM4685763 SRX8807834 GSM4685764 SRP273093 PRJNA647773 SRS7073895 GSM4685764 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685764 GSM4685764 GEO Accession GSM4685764 SRX8807835 GSM4685765 SRP273093 PRJNA647773 SRS7073896 GSM4685765 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685765 GSM4685765 GEO Accession GSM4685765 SRX8807836 GSM4685766 SRP273093 PRJNA647773 SRS7073897 GSM4685766 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685766 GSM4685766 GEO Accession GSM4685766 SRX8807837 GSM4685767 SRP273093 PRJNA647773 SRS7073898 GSM4685767 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685767 GSM4685767 GEO Accession GSM4685767 SRX8807838 GSM4685768 SRP273093 PRJNA647773 SRS7073899 GSM4685768 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685768 GSM4685768 GEO Accession GSM4685768 SRX8807839 GSM4685769 SRP273093 PRJNA647773 SRS7073900 GSM4685769 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685769 GSM4685769 GEO Accession GSM4685769 SRX8807840 GSM4685770 SRP273093 PRJNA647773 SRS7073901 GSM4685770 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685770 GSM4685770 GEO Accession GSM4685770 SRX8807841 GSM4685771 SRP273093 PRJNA647773 SRS7073902 GSM4685771 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685771 GSM4685771 GEO Accession GSM4685771 SRX8807842 GSM4685772 SRP273093 PRJNA647773 SRS7073903 GSM4685772 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685772 GSM4685772 GEO Accession GSM4685772 SRX8807843 GSM4685773 SRP273093 PRJNA647773 SRS7073904 GSM4685773 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685773 GSM4685773 GEO Accession GSM4685773 SRX8807844 GSM4685774 SRP273093 PRJNA647773 SRS7073905 GSM4685774 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685774 GSM4685774 GEO Accession GSM4685774 SRX8807845 GSM4685775 SRP273093 PRJNA647773 SRS7073906 GSM4685775 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685775 GSM4685775 GEO Accession GSM4685775 SRX8807846 GSM4685776 SRP273093 PRJNA647773 SRS7073907 GSM4685776 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685776 GSM4685776 GEO Accession GSM4685776 SRX8807847 GSM4685777 SRP273093 PRJNA647773 SRS7073908 GSM4685777 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685777 GSM4685777 GEO Accession GSM4685777 SRX8807848 GSM4685778 SRP273093 PRJNA647773 SRS7073909 GSM4685778 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685778 GSM4685778 GEO Accession GSM4685778 SRX8807849 GSM4685779 SRP273093 PRJNA647773 SRS7073910 GSM4685779 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685779 GSM4685779 GEO Accession GSM4685779 SRX8807850 GSM4685780 SRP273093 PRJNA647773 SRS7073911 GSM4685780 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685780 GSM4685780 GEO Accession GSM4685780 SRX8807851 GSM4685781 SRP273093 PRJNA647773 SRS7073912 GSM4685781 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685781 GSM4685781 GEO Accession GSM4685781 SRX8807852 GSM4685782 SRP273093 PRJNA647773 SRS7073913 GSM4685782 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685782 GSM4685782 GEO Accession GSM4685782 SRX8807853 GSM4685783 SRP273093 PRJNA647773 SRS7073914 GSM4685783 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685783 GSM4685783 GEO Accession GSM4685783 SRX8807854 GSM4685784 SRP273093 PRJNA647773 SRS7073915 GSM4685784 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685784 GSM4685784 GEO Accession GSM4685784 SRX8807855 GSM4685785 SRP273093 PRJNA647773 SRS7073916 GSM4685785 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685785 GSM4685785 GEO Accession GSM4685785 SRX8807856 GSM4685786 SRP273093 PRJNA647773 SRS7073917 GSM4685786 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685786 GSM4685786 GEO Accession GSM4685786 SRX8807857 GSM4685787 SRP273093 PRJNA647773 SRS7073918 GSM4685787 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685787 GSM4685787 GEO Accession GSM4685787 SRX8807858 GSM4685788 SRP273093 PRJNA647773 SRS7073919 GSM4685788 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685788 GSM4685788 GEO Accession GSM4685788 SRX8807859 GSM4685789 SRP273093 PRJNA647773 SRS7073920 GSM4685789 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685789 GSM4685789 GEO Accession GSM4685789 SRX8807860 GSM4685790 SRP273093 PRJNA647773 SRS7073921 GSM4685790 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685790 GSM4685790 GEO Accession GSM4685790 SRX8807861 GSM4685791 SRP273093 PRJNA647773 SRS7073922 GSM4685791 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685791 GSM4685791 GEO Accession GSM4685791 SRX8807862 GSM4685792 SRP273093 PRJNA647773 SRS7073923 GSM4685792 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685792 GSM4685792 GEO Accession GSM4685792 SRX8807863 GSM4685793 SRP273093 PRJNA647773 SRS7073924 GSM4685793 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685793 GSM4685793 GEO Accession GSM4685793 SRX8807864 GSM4685794 SRP273093 PRJNA647773 SRS7073925 GSM4685794 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685794 GSM4685794 GEO Accession GSM4685794 SRX8807865 GSM4685795 SRP273093 PRJNA647773 SRS7073926 GSM4685795 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685795 GSM4685795 GEO Accession GSM4685795 SRX8807866 GSM4685796 SRP273093 PRJNA647773 SRS7073927 GSM4685796 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685796 GSM4685796 GEO Accession GSM4685796 SRX8807867 GSM4685797 SRP273093 PRJNA647773 SRS7073928 GSM4685797 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685797 GSM4685797 GEO Accession GSM4685797 SRX8807868 GSM4685798 SRP273093 PRJNA647773 SRS7073929 GSM4685798 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685798 GSM4685798 GEO Accession GSM4685798 SRX8807869 GSM4685799 SRP273093 PRJNA647773 SRS7073930 GSM4685799 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685799 GSM4685799 GEO Accession GSM4685799 SRX8807870 GSM4685800 SRP273093 PRJNA647773 SRS7073931 GSM4685800 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685800 GSM4685800 GEO Accession GSM4685800 SRX8807871 GSM4685801 SRP273093 PRJNA647773 SRS7073932 GSM4685801 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685801 GSM4685801 GEO Accession GSM4685801 SRX8807872 GSM4685802 SRP273093 PRJNA647773 SRS7073933 GSM4685802 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685802 GSM4685802 GEO Accession GSM4685802 SRX8807873 GSM4685803 SRP273093 PRJNA647773 SRS7073934 GSM4685803 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685803 GSM4685803 GEO Accession GSM4685803 SRX8807874 GSM4685804 SRP273093 PRJNA647773 SRS7073935 GSM4685804 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685804 GSM4685804 GEO Accession GSM4685804 SRX8807875 GSM4685805 SRP273093 PRJNA647773 SRS7073936 GSM4685805 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685805 GSM4685805 GEO Accession GSM4685805 SRX8807876 GSM4685806 SRP273093 PRJNA647773 SRS7073937 GSM4685806 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685806 GSM4685806 GEO Accession GSM4685806 SRX8807877 GSM4685807 SRP273093 PRJNA647773 SRS7073938 GSM4685807 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685807 GSM4685807 GEO Accession GSM4685807 SRX8807878 GSM4685808 SRP273093 PRJNA647773 SRS7073939 GSM4685808 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685808 GSM4685808 GEO Accession GSM4685808 SRX8807879 GSM4685809 SRP273093 PRJNA647773 SRS7073940 GSM4685809 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685809 GSM4685809 GEO Accession GSM4685809 SRX8807880 GSM4685810 SRP273093 PRJNA647773 SRS7073941 GSM4685810 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685810 GSM4685810 GEO Accession GSM4685810 SRX8807881 GSM4685811 SRP273093 PRJNA647773 SRS7073942 GSM4685811 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685811 GSM4685811 GEO Accession GSM4685811 SRX8807882 GSM4685812 SRP273093 PRJNA647773 SRS7073943 GSM4685812 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685812 GSM4685812 GEO Accession GSM4685812 SRX8807883 GSM4685813 SRP273093 PRJNA647773 SRS7073944 GSM4685813 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685813 GSM4685813 GEO Accession GSM4685813 SRX8807884 GSM4685814 SRP273093 PRJNA647773 SRS7073946 GSM4685814 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685814 GSM4685814 GEO Accession GSM4685814 SRX8807885 GSM4685815 SRP273093 PRJNA647773 SRS7073945 GSM4685815 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685815 GSM4685815 GEO Accession GSM4685815 SRX8807886 GSM4685816 SRP273093 PRJNA647773 SRS7073947 GSM4685816 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685816 GSM4685816 GEO Accession GSM4685816 SRX8807887 GSM4685817 SRP273093 PRJNA647773 SRS7073949 GSM4685817 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685817 GSM4685817 GEO Accession GSM4685817 SRX8807888 GSM4685818 SRP273093 PRJNA647773 SRS7073948 GSM4685818 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685818 GSM4685818 GEO Accession GSM4685818 SRX8807889 GSM4685819 SRP273093 PRJNA647773 SRS7073950 GSM4685819 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685819 GSM4685819 GEO Accession GSM4685819 SRX8807890 GSM4685820 SRP273093 PRJNA647773 SRS7073951 GSM4685820 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685820 GSM4685820 GEO Accession GSM4685820 SRX8807891 GSM4685821 SRP273093 PRJNA647773 SRS7073952 GSM4685821 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685821 GSM4685821 GEO Accession GSM4685821 SRX8807892 GSM4685822 SRP273093 PRJNA647773 SRS7073953 GSM4685822 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685822 GSM4685822 GEO Accession GSM4685822 SRX8807893 GSM4685823 SRP273093 PRJNA647773 SRS7073954 GSM4685823 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685823 GSM4685823 GEO Accession GSM4685823 SRX8807894 GSM4685824 SRP273093 PRJNA647773 SRS7073955 GSM4685824 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685824 GSM4685824 GEO Accession GSM4685824 SRX8807895 GSM4685825 SRP273093 PRJNA647773 SRS7073956 GSM4685825 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685825 GSM4685825 GEO Accession GSM4685825 SRX8807896 GSM4685826 SRP273093 PRJNA647773 SRS7073957 GSM4685826 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685826 GSM4685826 GEO Accession GSM4685826 SRX8807897 GSM4685827 SRP273093 PRJNA647773 SRS7073958 GSM4685827 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685827 GSM4685827 GEO Accession GSM4685827 SRX8807898 GSM4685828 SRP273093 PRJNA647773 SRS7073959 GSM4685828 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685828 GSM4685828 GEO Accession GSM4685828 SRX8807899 GSM4685829 SRP273093 PRJNA647773 SRS7073960 GSM4685829 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685829 GSM4685829 GEO Accession GSM4685829 SRX8807900 GSM4685830 SRP273093 PRJNA647773 SRS7073962 GSM4685830 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685830 GSM4685830 GEO Accession GSM4685830 SRX8807901 GSM4685831 SRP273093 PRJNA647773 SRS7073961 GSM4685831 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685831 GSM4685831 GEO Accession GSM4685831 SRX8807902 GSM4685832 SRP273093 PRJNA647773 SRS7073963 GSM4685832 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685832 GSM4685832 GEO Accession GSM4685832 SRX8807903 GSM4685833 SRP273093 PRJNA647773 SRS7073964 GSM4685833 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685833 GSM4685833 GEO Accession GSM4685833 SRX8807904 GSM4685834 SRP273093 PRJNA647773 SRS7073965 GSM4685834 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685834 GSM4685834 GEO Accession GSM4685834 SRX8807905 GSM4685835 SRP273093 PRJNA647773 SRS7073966 GSM4685835 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685835 GSM4685835 GEO Accession GSM4685835 SRX8807906 GSM4685836 SRP273093 PRJNA647773 SRS7073967 GSM4685836 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685836 GSM4685836 GEO Accession GSM4685836 SRX8807907 GSM4685837 SRP273093 PRJNA647773 SRS7073968 GSM4685837 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685837 GSM4685837 GEO Accession GSM4685837 SRX8807908 GSM4685838 SRP273093 PRJNA647773 SRS7073969 GSM4685838 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685838 GSM4685838 GEO Accession GSM4685838 SRX8807909 GSM4685839 SRP273093 PRJNA647773 SRS7073970 GSM4685839 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685839 GSM4685839 GEO Accession GSM4685839 SRX8807910 GSM4685840 SRP273093 PRJNA647773 SRS7073971 GSM4685840 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685840 GSM4685840 GEO Accession GSM4685840 SRX8807911 GSM4685841 SRP273093 PRJNA647773 SRS7073972 GSM4685841 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685841 GSM4685841 GEO Accession GSM4685841 SRX8807912 GSM4685842 SRP273093 PRJNA647773 SRS7073973 GSM4685842 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685842 GSM4685842 GEO Accession GSM4685842 SRX8807913 GSM4685843 SRP273093 PRJNA647773 SRS7073974 GSM4685843 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685843 GSM4685843 GEO Accession GSM4685843 SRX8807914 GSM4685844 SRP273093 PRJNA647773 SRS7073975 GSM4685844 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685844 GSM4685844 GEO Accession GSM4685844 SRX8807915 GSM4685845 SRP273093 PRJNA647773 SRS7073976 GSM4685845 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685845 GSM4685845 GEO Accession GSM4685845 SRX8807916 GSM4685846 SRP273093 PRJNA647773 SRS7073977 GSM4685846 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685846 GSM4685846 GEO Accession GSM4685846 SRX8807917 GSM4685847 SRP273093 PRJNA647773 SRS7073978 GSM4685847 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685847 GSM4685847 GEO Accession GSM4685847 SRX8807918 GSM4685848 SRP273093 PRJNA647773 SRS7073979 GSM4685848 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685848 GSM4685848 GEO Accession GSM4685848 SRX8807919 GSM4685849 SRP273093 PRJNA647773 SRS7073980 GSM4685849 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685849 GSM4685849 GEO Accession GSM4685849 SRX8807920 GSM4685850 SRP273093 PRJNA647773 SRS7073981 GSM4685850 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685850 GSM4685850 GEO Accession GSM4685850 SRX8807921 GSM4685851 SRP273093 PRJNA647773 SRS7073982 GSM4685851 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685851 GSM4685851 GEO Accession GSM4685851 SRX8807922 GSM4685852 SRP273093 PRJNA647773 SRS7073983 GSM4685852 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685852 GSM4685852 GEO Accession GSM4685852 SRX8807923 GSM4685853 SRP273093 PRJNA647773 SRS7073984 GSM4685853 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685853 GSM4685853 GEO Accession GSM4685853 SRX8807924 GSM4685854 SRP273093 PRJNA647773 SRS7073985 GSM4685854 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685854 GSM4685854 GEO Accession GSM4685854 SRX8807925 GSM4685855 SRP273093 PRJNA647773 SRS7073986 GSM4685855 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685855 GSM4685855 GEO Accession GSM4685855 SRX8807926 GSM4685856 SRP273093 PRJNA647773 SRS7073987 GSM4685856 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685856 GSM4685856 GEO Accession GSM4685856 SRX8807927 GSM4685857 SRP273093 PRJNA647773 SRS7073988 GSM4685857 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685857 GSM4685857 GEO Accession GSM4685857 SRX8807928 GSM4685858 SRP273093 PRJNA647773 SRS7073989 GSM4685858 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685858 GSM4685858 GEO Accession GSM4685858 SRX8807929 GSM4685859 SRP273093 PRJNA647773 SRS7073990 GSM4685859 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685859 GSM4685859 GEO Accession GSM4685859 SRX8807930 GSM4685860 SRP273093 PRJNA647773 SRS7073991 GSM4685860 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685860 GSM4685860 GEO Accession GSM4685860 SRX8807931 GSM4685861 SRP273093 PRJNA647773 SRS7073992 GSM4685861 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685861 GSM4685861 GEO Accession GSM4685861 SRX8807932 GSM4685862 SRP273093 PRJNA647773 SRS7073993 GSM4685862 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685862 GSM4685862 GEO Accession GSM4685862 SRX8807933 GSM4685863 SRP273093 PRJNA647773 SRS7073994 GSM4685863 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685863 GSM4685863 GEO Accession GSM4685863 SRX8807934 GSM4685864 SRP273093 PRJNA647773 SRS7073995 GSM4685864 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685864 GSM4685864 GEO Accession GSM4685864 SRX8807935 GSM4685865 SRP273093 PRJNA647773 SRS7073996 GSM4685865 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685865 GSM4685865 GEO Accession GSM4685865 SRX8807936 GSM4685866 SRP273093 PRJNA647773 SRS7073997 GSM4685866 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685866 GSM4685866 GEO Accession GSM4685866 SRX8807937 GSM4685867 SRP273093 PRJNA647773 SRS7073998 GSM4685867 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685867 GSM4685867 GEO Accession GSM4685867 SRX8807938 GSM4685868 SRP273093 PRJNA647773 SRS7073999 GSM4685868 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685868 GSM4685868 GEO Accession GSM4685868 SRX8807939 GSM4685869 SRP273093 PRJNA647773 SRS7074000 GSM4685869 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685869 GSM4685869 GEO Accession GSM4685869 SRX8807940 GSM4685870 SRP273093 PRJNA647773 SRS7074001 GSM4685870 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685870 GSM4685870 GEO Accession GSM4685870 SRX8807941 GSM4685871 SRP273093 PRJNA647773 SRS7074002 GSM4685871 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685871 GSM4685871 GEO Accession GSM4685871 SRX8807942 GSM4685872 SRP273093 PRJNA647773 SRS7074003 GSM4685872 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685872 GSM4685872 GEO Accession GSM4685872 SRX8807943 GSM4685873 SRP273093 PRJNA647773 SRS7074004 GSM4685873 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685873 GSM4685873 GEO Accession GSM4685873 SRX8807944 GSM4685874 SRP273093 PRJNA647773 SRS7074005 GSM4685874 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685874 GSM4685874 GEO Accession GSM4685874 SRX8807945 GSM4685875 SRP273093 PRJNA647773 SRS7074006 GSM4685875 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685875 GSM4685875 GEO Accession GSM4685875 SRX8807946 GSM4685876 SRP273093 PRJNA647773 SRS7074007 GSM4685876 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685876 GSM4685876 GEO Accession GSM4685876 SRX8807947 GSM4685877 SRP273093 PRJNA647773 SRS7074008 GSM4685877 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685877 GSM4685877 GEO Accession GSM4685877 SRX8807948 GSM4685878 SRP273093 PRJNA647773 SRS7074009 GSM4685878 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685878 GSM4685878 GEO Accession GSM4685878 SRX8807949 GSM4685879 SRP273093 PRJNA647773 SRS7074010 GSM4685879 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685879 GSM4685879 GEO Accession GSM4685879 SRX8807950 GSM4685880 SRP273093 PRJNA647773 SRS7074011 GSM4685880 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685880 GSM4685880 GEO Accession GSM4685880 SRX8807951 GSM4685881 SRP273093 PRJNA647773 SRS7074012 GSM4685881 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685881 GSM4685881 GEO Accession GSM4685881 SRX8807952 GSM4685882 SRP273093 PRJNA647773 SRS7074013 GSM4685882 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685882 GSM4685882 GEO Accession GSM4685882 SRX8807953 GSM4685883 SRP273093 PRJNA647773 SRS7074014 GSM4685883 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685883 GSM4685883 GEO Accession GSM4685883 SRX8807954 GSM4685884 SRP273093 PRJNA647773 SRS7074015 GSM4685884 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685884 GSM4685884 GEO Accession GSM4685884 SRX8807955 GSM4685885 SRP273093 PRJNA647773 SRS7074016 GSM4685885 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685885 GSM4685885 GEO Accession GSM4685885 SRX8807956 GSM4685886 SRP273093 PRJNA647773 SRS7074017 GSM4685886 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685886 GSM4685886 GEO Accession GSM4685886 SRX8807957 GSM4685887 SRP273093 PRJNA647773 SRS7074018 GSM4685887 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685887 GSM4685887 GEO Accession GSM4685887 SRX8807958 GSM4685888 SRP273093 PRJNA647773 SRS7074019 GSM4685888 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685888 GSM4685888 GEO Accession GSM4685888 SRX8807959 GSM4685889 SRP273093 PRJNA647773 SRS7074020 GSM4685889 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685889 GSM4685889 GEO Accession GSM4685889 SRX8807960 GSM4685890 SRP273093 PRJNA647773 SRS7074021 GSM4685890 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685890 GSM4685890 GEO Accession GSM4685890 SRX8807961 GSM4685891 SRP273093 PRJNA647773 SRS7074022 GSM4685891 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685891 GSM4685891 GEO Accession GSM4685891 SRX8807962 GSM4685892 SRP273093 PRJNA647773 SRS7074023 GSM4685892 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685892 GSM4685892 GEO Accession GSM4685892 SRX8807963 GSM4685893 SRP273093 PRJNA647773 SRS7074024 GSM4685893 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685893 GSM4685893 GEO Accession GSM4685893 SRX8807964 GSM4685894 SRP273093 PRJNA647773 SRS7074025 GSM4685894 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685894 GSM4685894 GEO Accession GSM4685894 SRX8807965 GSM4685895 SRP273093 PRJNA647773 SRS7074026 GSM4685895 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685895 GSM4685895 GEO Accession GSM4685895 SRX8807966 GSM4685896 SRP273093 PRJNA647773 SRS7074027 GSM4685896 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685896 GSM4685896 GEO Accession GSM4685896 SRX8807967 GSM4685897 SRP273093 PRJNA647773 SRS7074028 GSM4685897 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685897 GSM4685897 GEO Accession GSM4685897 SRX8807968 GSM4685898 SRP273093 PRJNA647773 SRS7074029 GSM4685898 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685898 GSM4685898 GEO Accession GSM4685898 SRX8807969 GSM4685899 SRP273093 PRJNA647773 SRS7074030 GSM4685899 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685899 GSM4685899 GEO Accession GSM4685899 SRX8807970 GSM4685900 SRP273093 PRJNA647773 SRS7074031 GSM4685900 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685900 GSM4685900 GEO Accession GSM4685900 SRX8807971 GSM4685901 SRP273093 PRJNA647773 SRS7074032 GSM4685901 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685901 GSM4685901 GEO Accession GSM4685901 SRX8807972 GSM4685902 SRP273093 PRJNA647773 SRS7074033 GSM4685902 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685902 GSM4685902 GEO Accession GSM4685902 SRX8807973 GSM4685903 SRP273093 PRJNA647773 SRS7074034 GSM4685903 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685903 GSM4685903 GEO Accession GSM4685903 SRX8807974 GSM4685904 SRP273093 PRJNA647773 SRS7074035 GSM4685904 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685904 GSM4685904 GEO Accession GSM4685904 SRX8807975 GSM4685905 SRP273093 PRJNA647773 SRS7074036 GSM4685905 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685905 GSM4685905 GEO Accession GSM4685905 SRX8807976 GSM4685906 SRP273093 PRJNA647773 SRS7074037 GSM4685906 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685906 GSM4685906 GEO Accession GSM4685906 SRX8807977 GSM4685907 SRP273093 PRJNA647773 SRS7074038 GSM4685907 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685907 GSM4685907 GEO Accession GSM4685907 SRX8807978 GSM4685908 SRP273093 PRJNA647773 SRS7074039 GSM4685908 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685908 GSM4685908 GEO Accession GSM4685908 SRX8807979 GSM4685909 SRP273093 PRJNA647773 SRS7074040 GSM4685909 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685909 GSM4685909 GEO Accession GSM4685909 SRX8807980 GSM4685910 SRP273093 PRJNA647773 SRS7074041 GSM4685910 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685910 GSM4685910 GEO Accession GSM4685910 SRX8807981 GSM4685911 SRP273093 PRJNA647773 SRS7074042 GSM4685911 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685911 GSM4685911 GEO Accession GSM4685911 SRX8807982 GSM4685912 SRP273093 PRJNA647773 SRS7074043 GSM4685912 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685912 GSM4685912 GEO Accession GSM4685912 SRX8807983 GSM4685913 SRP273093 PRJNA647773 SRS7074044 GSM4685913 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685913 GSM4685913 GEO Accession GSM4685913 SRX8807984 GSM4685914 SRP273093 PRJNA647773 SRS7074045 GSM4685914 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685914 GSM4685914 GEO Accession GSM4685914 SRX8807985 GSM4685915 SRP273093 PRJNA647773 SRS7074046 GSM4685915 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685915 GSM4685915 GEO Accession GSM4685915 SRX8807986 GSM4685916 SRP273093 PRJNA647773 SRS7074047 GSM4685916 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685916 GSM4685916 GEO Accession GSM4685916 SRX8807987 GSM4685917 SRP273093 PRJNA647773 SRS7074048 GSM4685917 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685917 GSM4685917 GEO Accession GSM4685917 SRX8807988 GSM4685918 SRP273093 PRJNA647773 SRS7074049 GSM4685918 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685918 GSM4685918 GEO Accession GSM4685918 SRX8807989 GSM4685919 SRP273093 PRJNA647773 SRS7074050 GSM4685919 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685919 GSM4685919 GEO Accession GSM4685919 SRX8807990 GSM4685920 SRP273093 PRJNA647773 SRS7074051 GSM4685920 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685920 GSM4685920 GEO Accession GSM4685920 SRX8807991 GSM4685921 SRP273093 PRJNA647773 SRS7074052 GSM4685921 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685921 GSM4685921 GEO Accession GSM4685921 SRX8807992 GSM4685922 SRP273093 PRJNA647773 SRS7074053 GSM4685922 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685922 GSM4685922 GEO Accession GSM4685922 SRX8807993 GSM4685923 SRP273093 PRJNA647773 SRS7074054 GSM4685923 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685923 GSM4685923 GEO Accession GSM4685923 SRX8807994 GSM4685924 SRP273093 PRJNA647773 SRS7074055 GSM4685924 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685924 GSM4685924 GEO Accession GSM4685924 SRX8807995 GSM4685925 SRP273093 PRJNA647773 SRS7074056 GSM4685925 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685925 GSM4685925 GEO Accession GSM4685925 SRX8807996 GSM4685926 SRP273093 PRJNA647773 SRS7074057 GSM4685926 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685926 GSM4685926 GEO Accession GSM4685926 SRX8807997 GSM4685927 SRP273093 PRJNA647773 SRS7074058 GSM4685927 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685927 GSM4685927 GEO Accession GSM4685927 SRX8807998 GSM4685928 SRP273093 PRJNA647773 SRS7074059 GSM4685928 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685928 GSM4685928 GEO Accession GSM4685928 SRX8807999 GSM4685929 SRP273093 PRJNA647773 SRS7074060 GSM4685929 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685929 GSM4685929 GEO Accession GSM4685929 SRX8808000 GSM4685930 SRP273093 PRJNA647773 SRS7074061 GSM4685930 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685930 GSM4685930 GEO Accession GSM4685930 SRX8808001 GSM4685931 SRP273093 PRJNA647773 SRS7074062 GSM4685931 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685931 GSM4685931 GEO Accession GSM4685931 SRX8808002 GSM4685932 SRP273093 PRJNA647773 SRS7074063 GSM4685932 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685932 GSM4685932 GEO Accession GSM4685932 SRX8808003 GSM4685933 SRP273093 PRJNA647773 SRS7074064 GSM4685933 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685933 GSM4685933 GEO Accession GSM4685933 SRX8808004 GSM4685934 SRP273093 PRJNA647773 SRS7074065 GSM4685934 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685934 GSM4685934 GEO Accession GSM4685934 SRX8808005 GSM4685935 SRP273093 PRJNA647773 SRS7074066 GSM4685935 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685935 GSM4685935 GEO Accession GSM4685935 SRX8808006 GSM4685936 SRP273093 PRJNA647773 SRS7074067 GSM4685936 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685936 GSM4685936 GEO Accession GSM4685936 SRX8808007 GSM4685937 SRP273093 PRJNA647773 SRS7074068 GSM4685937 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685937 GSM4685937 GEO Accession GSM4685937 SRX8808008 GSM4685938 SRP273093 PRJNA647773 SRS7074071 GSM4685938 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685938 GSM4685938 GEO Accession GSM4685938 SRX8808009 GSM4685939 SRP273093 PRJNA647773 SRS7074069 GSM4685939 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685939 GSM4685939 GEO Accession GSM4685939 SRX8808010 GSM4685940 SRP273093 PRJNA647773 SRS7074070 GSM4685940 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685940 GSM4685940 GEO Accession GSM4685940 SRX8808011 GSM4685941 SRP273093 PRJNA647773 SRS7074072 GSM4685941 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685941 GSM4685941 GEO Accession GSM4685941 SRX8808012 GSM4685942 SRP273093 PRJNA647773 SRS7074075 GSM4685942 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685942 GSM4685942 GEO Accession GSM4685942 SRX8808013 GSM4685943 SRP273093 PRJNA647773 SRS7074073 GSM4685943 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685943 GSM4685943 GEO Accession GSM4685943 SRX8808014 GSM4685944 SRP273093 PRJNA647773 SRS7074074 GSM4685944 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685944 GSM4685944 GEO Accession GSM4685944 SRX8808015 GSM4685945 SRP273093 PRJNA647773 SRS7074076 GSM4685945 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685945 GSM4685945 GEO Accession GSM4685945 SRX8808016 GSM4685946 SRP273093 PRJNA647773 SRS7074077 GSM4685946 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685946 GSM4685946 GEO Accession GSM4685946 SRX8808017 GSM4685947 SRP273093 PRJNA647773 SRS7074078 GSM4685947 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685947 GSM4685947 GEO Accession GSM4685947 SRX8808018 GSM4685948 SRP273093 PRJNA647773 SRS7074080 GSM4685948 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685948 GSM4685948 GEO Accession GSM4685948 SRX8808019 GSM4685949 SRP273093 PRJNA647773 SRS7074079 GSM4685949 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685949 GSM4685949 GEO Accession GSM4685949 SRX8808020 GSM4685950 SRP273093 PRJNA647773 SRS7074082 GSM4685950 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685950 GSM4685950 GEO Accession GSM4685950 SRX8808021 GSM4685951 SRP273093 PRJNA647773 SRS7074081 GSM4685951 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685951 GSM4685951 GEO Accession GSM4685951 SRX8808022 GSM4685952 SRP273093 PRJNA647773 SRS7074084 GSM4685952 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685952 GSM4685952 GEO Accession GSM4685952 SRX8808023 GSM4685953 SRP273093 PRJNA647773 SRS7074083 GSM4685953 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685953 GSM4685953 GEO Accession GSM4685953 SRX8808024 GSM4685954 SRP273093 PRJNA647773 SRS7074086 GSM4685954 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685954 GSM4685954 GEO Accession GSM4685954 SRX8808025 GSM4685955 SRP273093 PRJNA647773 SRS7074085 GSM4685955 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685955 GSM4685955 GEO Accession GSM4685955 SRX8808026 GSM4685956 SRP273093 PRJNA647773 SRS7074087 GSM4685956 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685956 GSM4685956 GEO Accession GSM4685956 SRX8808027 GSM4685957 SRP273093 PRJNA647773 SRS7074088 GSM4685957 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685957 GSM4685957 GEO Accession GSM4685957 SRX8808028 GSM4685958 SRP273093 PRJNA647773 SRS7074089 GSM4685958 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685958 GSM4685958 GEO Accession GSM4685958 SRX8808029 GSM4685959 SRP273093 PRJNA647773 SRS7074090 GSM4685959 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685959 GSM4685959 GEO Accession GSM4685959 SRX8808030 GSM4685960 SRP273093 PRJNA647773 SRS7074091 GSM4685960 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685960 GSM4685960 GEO Accession GSM4685960 SRX8808031 GSM4685961 SRP273093 PRJNA647773 SRS7074092 GSM4685961 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685961 GSM4685961 GEO Accession GSM4685961 SRX8808032 GSM4685962 SRP273093 PRJNA647773 SRS7074093 GSM4685962 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685962 GSM4685962 GEO Accession GSM4685962 SRX8808033 GSM4685963 SRP273093 PRJNA647773 SRS7074094 GSM4685963 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685963 GSM4685963 GEO Accession GSM4685963 SRX8808034 GSM4685964 SRP273093 PRJNA647773 SRS7074095 GSM4685964 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685964 GSM4685964 GEO Accession GSM4685964 SRX8808035 GSM4685965 SRP273093 PRJNA647773 SRS7074096 GSM4685965 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685965 GSM4685965 GEO Accession GSM4685965 SRX8808036 GSM4685966 SRP273093 PRJNA647773 SRS7074097 GSM4685966 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685966 GSM4685966 GEO Accession GSM4685966 SRX8808037 GSM4685967 SRP273093 PRJNA647773 SRS7074098 GSM4685967 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685967 GSM4685967 GEO Accession GSM4685967 SRX8808038 GSM4685968 SRP273093 PRJNA647773 SRS7074099 GSM4685968 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685968 GSM4685968 GEO Accession GSM4685968 SRX8808039 GSM4685969 SRP273093 PRJNA647773 SRS7074100 GSM4685969 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685969 GSM4685969 GEO Accession GSM4685969 SRX8808040 GSM4685970 SRP273093 PRJNA647773 SRS7074101 GSM4685970 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685970 GSM4685970 GEO Accession GSM4685970 SRX8808041 GSM4685971 SRP273093 PRJNA647773 SRS7074102 GSM4685971 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685971 GSM4685971 GEO Accession GSM4685971 SRX8808042 GSM4685972 SRP273093 PRJNA647773 SRS7074103 GSM4685972 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685972 GSM4685972 GEO Accession GSM4685972 SRX8808043 GSM4685973 SRP273093 PRJNA647773 SRS7074104 GSM4685973 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685973 GSM4685973 GEO Accession GSM4685973 SRX8808044 GSM4685974 SRP273093 PRJNA647773 SRS7074105 GSM4685974 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685974 GSM4685974 GEO Accession GSM4685974 SRX8808045 GSM4685975 SRP273093 PRJNA647773 SRS7074106 GSM4685975 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685975 GSM4685975 GEO Accession GSM4685975 SRX8808046 GSM4685976 SRP273093 PRJNA647773 SRS7074107 GSM4685976 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685976 GSM4685976 GEO Accession GSM4685976 SRX8808047 GSM4685977 SRP273093 PRJNA647773 SRS7074108 GSM4685977 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685977 GSM4685977 GEO Accession GSM4685977 SRX8808048 GSM4685978 SRP273093 PRJNA647773 SRS7074109 GSM4685978 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685978 GSM4685978 GEO Accession GSM4685978 SRX8808049 GSM4685979 SRP273093 PRJNA647773 SRS7074110 GSM4685979 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685979 GSM4685979 GEO Accession GSM4685979 SRX8808050 GSM4685980 SRP273093 PRJNA647773 SRS7074111 GSM4685980 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685980 GSM4685980 GEO Accession GSM4685980 SRX8808051 GSM4685981 SRP273093 PRJNA647773 SRS7074112 GSM4685981 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685981 GSM4685981 GEO Accession GSM4685981 SRX8808052 GSM4685982 SRP273093 PRJNA647773 SRS7074113 GSM4685982 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685982 GSM4685982 GEO Accession GSM4685982 SRX8808053 GSM4685983 SRP273093 PRJNA647773 SRS7074114 GSM4685983 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685983 GSM4685983 GEO Accession GSM4685983 SRX8808054 GSM4685984 SRP273093 PRJNA647773 SRS7074115 GSM4685984 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685984 GSM4685984 GEO Accession GSM4685984 SRX8808055 GSM4685985 SRP273093 PRJNA647773 SRS7074116 GSM4685985 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685985 GSM4685985 GEO Accession GSM4685985 SRX8808056 GSM4685986 SRP273093 PRJNA647773 SRS7074117 GSM4685986 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685986 GSM4685986 GEO Accession GSM4685986 SRX8808057 GSM4685987 SRP273093 PRJNA647773 SRS7074118 GSM4685987 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685987 GSM4685987 GEO Accession GSM4685987 SRX8808058 GSM4685988 SRP273093 PRJNA647773 SRS7074119 GSM4685988 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685988 GSM4685988 GEO Accession GSM4685988 SRX8808059 GSM4685989 SRP273093 PRJNA647773 SRS7074120 GSM4685989 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685989 GSM4685989 GEO Accession GSM4685989 SRX8808060 GSM4685990 SRP273093 PRJNA647773 SRS7074121 GSM4685990 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685990 GSM4685990 GEO Accession GSM4685990 SRX8808061 GSM4685991 SRP273093 PRJNA647773 SRS7074122 GSM4685991 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685991 GSM4685991 GEO Accession GSM4685991 SRX8808062 GSM4685992 SRP273093 PRJNA647773 SRS7074123 GSM4685992 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685992 GSM4685992 GEO Accession GSM4685992 SRX8808063 GSM4685993 SRP273093 PRJNA647773 SRS7074124 GSM4685993 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685993 GSM4685993 GEO Accession GSM4685993 SRX8808064 GSM4685994 SRP273093 PRJNA647773 SRS7074125 GSM4685994 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685994 GSM4685994 GEO Accession GSM4685994 SRX8808065 GSM4685995 SRP273093 PRJNA647773 SRS7074126 GSM4685995 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685995 GSM4685995 GEO Accession GSM4685995 SRX8808066 GSM4685996 SRP273093 PRJNA647773 SRS7074127 GSM4685996 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685996 GSM4685996 GEO Accession GSM4685996 SRX8808067 GSM4685997 SRP273093 PRJNA647773 SRS7074129 GSM4685997 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685997 GSM4685997 GEO Accession GSM4685997 SRX8808068 GSM4685998 SRP273093 PRJNA647773 SRS7074128 GSM4685998 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685998 GSM4685998 GEO Accession GSM4685998 SRX8808069 GSM4685999 SRP273093 PRJNA647773 SRS7074130 GSM4685999 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304685999 GSM4685999 GEO Accession GSM4685999 SRX8808070 GSM4686000 SRP273093 PRJNA647773 SRS7074131 GSM4686000 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686000 GSM4686000 GEO Accession GSM4686000 SRX8808071 GSM4686001 SRP273093 PRJNA647773 SRS7074132 GSM4686001 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686001 GSM4686001 GEO Accession GSM4686001 SRX8808072 GSM4686002 SRP273093 PRJNA647773 SRS7074133 GSM4686002 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686002 GSM4686002 GEO Accession GSM4686002 SRX8808073 GSM4686003 SRP273093 PRJNA647773 SRS7074134 GSM4686003 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686003 GSM4686003 GEO Accession GSM4686003 SRX8808074 GSM4686004 SRP273093 PRJNA647773 SRS7074135 GSM4686004 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686004 GSM4686004 GEO Accession GSM4686004 SRX8808075 GSM4686005 SRP273093 PRJNA647773 SRS7074136 GSM4686005 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686005 GSM4686005 GEO Accession GSM4686005 SRX8808076 GSM4686006 SRP273093 PRJNA647773 SRS7074137 GSM4686006 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686006 GSM4686006 GEO Accession GSM4686006 SRX8808077 GSM4686007 SRP273093 PRJNA647773 SRS7074138 GSM4686007 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686007 GSM4686007 GEO Accession GSM4686007 SRX8808078 GSM4686008 SRP273093 PRJNA647773 SRS7074139 GSM4686008 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686008 GSM4686008 GEO Accession GSM4686008 SRX8808079 GSM4686009 SRP273093 PRJNA647773 SRS7074140 GSM4686009 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686009 GSM4686009 GEO Accession GSM4686009 SRX8808080 GSM4686010 SRP273093 PRJNA647773 SRS7074141 GSM4686010 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686010 GSM4686010 GEO Accession GSM4686010 SRX8808081 GSM4686011 SRP273093 PRJNA647773 SRS7074142 GSM4686011 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686011 GSM4686011 GEO Accession GSM4686011 SRX8808082 GSM4686012 SRP273093 PRJNA647773 SRS7074143 GSM4686012 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686012 GSM4686012 GEO Accession GSM4686012 SRX8808083 GSM4686013 SRP273093 PRJNA647773 SRS7074144 GSM4686013 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686013 GSM4686013 GEO Accession GSM4686013 SRX8808084 GSM4686014 SRP273093 PRJNA647773 SRS7074145 GSM4686014 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686014 GSM4686014 GEO Accession GSM4686014 SRX8808085 GSM4686015 SRP273093 PRJNA647773 SRS7074146 GSM4686015 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686015 GSM4686015 GEO Accession GSM4686015 SRX8808086 GSM4686016 SRP273093 PRJNA647773 SRS7074148 GSM4686016 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686016 GSM4686016 GEO Accession GSM4686016 SRX8808087 GSM4686017 SRP273093 PRJNA647773 SRS7074147 GSM4686017 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686017 GSM4686017 GEO Accession GSM4686017 SRX8808088 GSM4686018 SRP273093 PRJNA647773 SRS7074149 GSM4686018 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686018 GSM4686018 GEO Accession GSM4686018 SRX8808089 GSM4686019 SRP273093 PRJNA647773 SRS7074150 GSM4686019 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686019 GSM4686019 GEO Accession GSM4686019 SRX8808090 GSM4686020 SRP273093 PRJNA647773 SRS7074151 GSM4686020 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686020 GSM4686020 GEO Accession GSM4686020 SRX8808091 GSM4686021 SRP273093 PRJNA647773 SRS7074152 GSM4686021 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686021 GSM4686021 GEO Accession GSM4686021 SRX8808092 GSM4686022 SRP273093 PRJNA647773 SRS7074153 GSM4686022 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686022 GSM4686022 GEO Accession GSM4686022 SRX8808093 GSM4686023 SRP273093 PRJNA647773 SRS7074154 GSM4686023 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686023 GSM4686023 GEO Accession GSM4686023 SRX8808094 GSM4686024 SRP273093 PRJNA647773 SRS7074155 GSM4686024 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686024 GSM4686024 GEO Accession GSM4686024 SRX8808095 GSM4686025 SRP273093 PRJNA647773 SRS7074156 GSM4686025 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686025 GSM4686025 GEO Accession GSM4686025 SRX8808096 GSM4686026 SRP273093 PRJNA647773 SRS7074157 GSM4686026 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686026 GSM4686026 GEO Accession GSM4686026 SRX8808097 GSM4686027 SRP273093 PRJNA647773 SRS7074158 GSM4686027 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686027 GSM4686027 GEO Accession GSM4686027 SRX8808098 GSM4686028 SRP273093 PRJNA647773 SRS7074159 GSM4686028 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686028 GSM4686028 GEO Accession GSM4686028 SRX8808099 GSM4686029 SRP273093 PRJNA647773 SRS7074160 GSM4686029 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686029 GSM4686029 GEO Accession GSM4686029 SRX8808100 GSM4686030 SRP273093 PRJNA647773 SRS7074161 GSM4686030 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686030 GSM4686030 GEO Accession GSM4686030 SRX8808101 GSM4686031 SRP273093 PRJNA647773 SRS7074162 GSM4686031 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686031 GSM4686031 GEO Accession GSM4686031 SRX8808102 GSM4686032 SRP273093 PRJNA647773 SRS7074163 GSM4686032 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686032 GSM4686032 GEO Accession GSM4686032 SRX8808103 GSM4686033 SRP273093 PRJNA647773 SRS7074164 GSM4686033 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686033 GSM4686033 GEO Accession GSM4686033 SRX8808104 GSM4686034 SRP273093 PRJNA647773 SRS7074165 GSM4686034 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686034 GSM4686034 GEO Accession GSM4686034 SRX8808105 GSM4686035 SRP273093 PRJNA647773 SRS7074166 GSM4686035 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686035 GSM4686035 GEO Accession GSM4686035 SRX8808106 GSM4686036 SRP273093 PRJNA647773 SRS7074167 GSM4686036 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686036 GSM4686036 GEO Accession GSM4686036 SRX8808107 GSM4686037 SRP273093 PRJNA647773 SRS7074168 GSM4686037 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686037 GSM4686037 GEO Accession GSM4686037 SRX8808108 GSM4686038 SRP273093 PRJNA647773 SRS7074169 GSM4686038 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686038 GSM4686038 GEO Accession GSM4686038 SRX8808109 GSM4686039 SRP273093 PRJNA647773 SRS7074170 GSM4686039 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686039 GSM4686039 GEO Accession GSM4686039 SRX8808110 GSM4686040 SRP273093 PRJNA647773 SRS7074171 GSM4686040 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686040 GSM4686040 GEO Accession GSM4686040 SRX8808111 GSM4686041 SRP273093 PRJNA647773 SRS7074172 GSM4686041 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686041 GSM4686041 GEO Accession GSM4686041 SRX8808112 GSM4686042 SRP273093 PRJNA647773 SRS7074173 GSM4686042 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686042 GSM4686042 GEO Accession GSM4686042 SRX8808113 GSM4686043 SRP273093 PRJNA647773 SRS7074174 GSM4686043 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686043 GSM4686043 GEO Accession GSM4686043 SRX8808114 GSM4686044 SRP273093 PRJNA647773 SRS7074175 GSM4686044 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686044 GSM4686044 GEO Accession GSM4686044 SRX8808115 GSM4686045 SRP273093 PRJNA647773 SRS7074176 GSM4686045 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686045 GSM4686045 GEO Accession GSM4686045 SRX8808116 GSM4686046 SRP273093 PRJNA647773 SRS7074177 GSM4686046 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686046 GSM4686046 GEO Accession GSM4686046 SRX8808117 GSM4686047 SRP273093 PRJNA647773 SRS7074178 GSM4686047 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686047 GSM4686047 GEO Accession GSM4686047 SRX8808118 GSM4686048 SRP273093 PRJNA647773 SRS7074179 GSM4686048 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686048 GSM4686048 GEO Accession GSM4686048 SRX8808119 GSM4686049 SRP273093 PRJNA647773 SRS7074180 GSM4686049 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686049 GSM4686049 GEO Accession GSM4686049 SRX8808120 GSM4686050 SRP273093 PRJNA647773 SRS7074181 GSM4686050 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686050 GSM4686050 GEO Accession GSM4686050 SRX8808121 GSM4686051 SRP273093 PRJNA647773 SRS7074183 GSM4686051 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686051 GSM4686051 GEO Accession GSM4686051 SRX8808122 GSM4686052 SRP273093 PRJNA647773 SRS7074182 GSM4686052 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686052 GSM4686052 GEO Accession GSM4686052 SRX8808123 GSM4686053 SRP273093 PRJNA647773 SRS7074184 GSM4686053 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686053 GSM4686053 GEO Accession GSM4686053 SRX8808124 GSM4686054 SRP273093 PRJNA647773 SRS7074185 GSM4686054 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686054 GSM4686054 GEO Accession GSM4686054 SRX8808125 GSM4686055 SRP273093 PRJNA647773 SRS7074186 GSM4686055 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686055 GSM4686055 GEO Accession GSM4686055 SRX8808126 GSM4686056 SRP273093 PRJNA647773 SRS7074187 GSM4686056 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686056 GSM4686056 GEO Accession GSM4686056 SRX8808127 GSM4686057 SRP273093 PRJNA647773 SRS7074188 GSM4686057 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686057 GSM4686057 GEO Accession GSM4686057 SRX8808128 GSM4686058 SRP273093 PRJNA647773 SRS7074189 GSM4686058 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686058 GSM4686058 GEO Accession GSM4686058 SRX8808129 GSM4686059 SRP273093 PRJNA647773 SRS7074190 GSM4686059 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686059 GSM4686059 GEO Accession GSM4686059 SRX8808130 GSM4686060 SRP273093 PRJNA647773 SRS7074191 GSM4686060 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686060 GSM4686060 GEO Accession GSM4686060 SRX8808131 GSM4686061 SRP273093 PRJNA647773 SRS7074192 GSM4686061 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686061 GSM4686061 GEO Accession GSM4686061 SRX8808132 GSM4686062 SRP273093 PRJNA647773 SRS7074193 GSM4686062 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686062 GSM4686062 GEO Accession GSM4686062 SRX8808133 GSM4686063 SRP273093 PRJNA647773 SRS7074194 GSM4686063 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686063 GSM4686063 GEO Accession GSM4686063 SRX8808134 GSM4686064 SRP273093 PRJNA647773 SRS7074195 GSM4686064 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686064 GSM4686064 GEO Accession GSM4686064 SRX8808135 GSM4686065 SRP273093 PRJNA647773 SRS7074196 GSM4686065 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686065 GSM4686065 GEO Accession GSM4686065 SRX8808136 GSM4686066 SRP273093 PRJNA647773 SRS7074197 GSM4686066 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686066 GSM4686066 GEO Accession GSM4686066 SRX8808137 GSM4686067 SRP273093 PRJNA647773 SRS7074198 GSM4686067 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686067 GSM4686067 GEO Accession GSM4686067 SRX8808138 GSM4686068 SRP273093 PRJNA647773 SRS7074199 GSM4686068 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686068 GSM4686068 GEO Accession GSM4686068 SRX8808139 GSM4686069 SRP273093 PRJNA647773 SRS7074202 GSM4686069 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686069 GSM4686069 GEO Accession GSM4686069 SRX8808140 GSM4686070 SRP273093 PRJNA647773 SRS7074201 GSM4686070 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686070 GSM4686070 GEO Accession GSM4686070 SRX8808141 GSM4686071 SRP273093 PRJNA647773 SRS7074200 GSM4686071 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686071 GSM4686071 GEO Accession GSM4686071 SRX8808142 GSM4686072 SRP273093 PRJNA647773 SRS7074203 GSM4686072 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686072 GSM4686072 GEO Accession GSM4686072 SRX8808143 GSM4686073 SRP273093 PRJNA647773 SRS7074204 GSM4686073 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686073 GSM4686073 GEO Accession GSM4686073 SRX8808144 GSM4686074 SRP273093 PRJNA647773 SRS7074205 GSM4686074 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686074 GSM4686074 GEO Accession GSM4686074 SRX8808145 GSM4686075 SRP273093 PRJNA647773 SRS7074207 GSM4686075 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686075 GSM4686075 GEO Accession GSM4686075 SRX8808146 GSM4686076 SRP273093 PRJNA647773 SRS7074206 GSM4686076 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686076 GSM4686076 GEO Accession GSM4686076 SRX8808147 GSM4686077 SRP273093 PRJNA647773 SRS7074208 GSM4686077 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686077 GSM4686077 GEO Accession GSM4686077 SRX8808148 GSM4686078 SRP273093 PRJNA647773 SRS7074209 GSM4686078 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686078 GSM4686078 GEO Accession GSM4686078 SRX8808149 GSM4686079 SRP273093 PRJNA647773 SRS7074210 GSM4686079 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686079 GSM4686079 GEO Accession GSM4686079 SRX8808150 GSM4686080 SRP273093 PRJNA647773 SRS7074211 GSM4686080 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686080 GSM4686080 GEO Accession GSM4686080 SRX8808151 GSM4686081 SRP273093 PRJNA647773 SRS7074212 GSM4686081 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686081 GSM4686081 GEO Accession GSM4686081 SRX8808152 GSM4686082 SRP273093 PRJNA647773 SRS7074213 GSM4686082 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686082 GSM4686082 GEO Accession GSM4686082 SRX8808153 GSM4686083 SRP273093 PRJNA647773 SRS7074214 GSM4686083 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686083 GSM4686083 GEO Accession GSM4686083 SRX8808154 GSM4686084 SRP273093 PRJNA647773 SRS7074216 GSM4686084 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686084 GSM4686084 GEO Accession GSM4686084 SRX8808155 GSM4686085 SRP273093 PRJNA647773 SRS7074215 GSM4686085 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686085 GSM4686085 GEO Accession GSM4686085 SRX8808156 GSM4686086 SRP273093 PRJNA647773 SRS7074217 GSM4686086 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686086 GSM4686086 GEO Accession GSM4686086 SRX8808157 GSM4686087 SRP273093 PRJNA647773 SRS7074218 GSM4686087 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686087 GSM4686087 GEO Accession GSM4686087 SRX8808158 GSM4686088 SRP273093 PRJNA647773 SRS7074219 GSM4686088 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686088 GSM4686088 GEO Accession GSM4686088 SRX8808159 GSM4686089 SRP273093 PRJNA647773 SRS7074220 GSM4686089 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686089 GSM4686089 GEO Accession GSM4686089 SRX8808160 GSM4686090 SRP273093 PRJNA647773 SRS7074221 GSM4686090 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686090 GSM4686090 GEO Accession GSM4686090 SRX8808161 GSM4686091 SRP273093 PRJNA647773 SRS7074222 GSM4686091 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686091 GSM4686091 GEO Accession GSM4686091 SRX8808162 GSM4686092 SRP273093 PRJNA647773 SRS7074223 GSM4686092 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686092 GSM4686092 GEO Accession GSM4686092 SRX8808163 GSM4686093 SRP273093 PRJNA647773 SRS7074224 GSM4686093 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686093 GSM4686093 GEO Accession GSM4686093 SRX8808164 GSM4686094 SRP273093 PRJNA647773 SRS7074225 GSM4686094 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686094 GSM4686094 GEO Accession GSM4686094 SRX8808165 GSM4686095 SRP273093 PRJNA647773 SRS7074226 GSM4686095 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686095 GSM4686095 GEO Accession GSM4686095 SRX8808166 GSM4686096 SRP273093 PRJNA647773 SRS7074227 GSM4686096 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686096 GSM4686096 GEO Accession GSM4686096 SRX8808167 GSM4686097 SRP273093 PRJNA647773 SRS7074228 GSM4686097 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686097 GSM4686097 GEO Accession GSM4686097 SRX8808168 GSM4686098 SRP273093 PRJNA647773 SRS7074229 GSM4686098 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686098 GSM4686098 GEO Accession GSM4686098 SRX8808169 GSM4686099 SRP273093 PRJNA647773 SRS7074230 GSM4686099 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686099 GSM4686099 GEO Accession GSM4686099 SRX8808170 GSM4686100 SRP273093 PRJNA647773 SRS7074231 GSM4686100 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686100 GSM4686100 GEO Accession GSM4686100 SRX8808171 GSM4686101 SRP273093 PRJNA647773 SRS7074232 GSM4686101 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686101 GSM4686101 GEO Accession GSM4686101 SRX8808172 GSM4686102 SRP273093 PRJNA647773 SRS7074233 GSM4686102 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686102 GSM4686102 GEO Accession GSM4686102 SRX8808173 GSM4686103 SRP273093 PRJNA647773 SRS7074234 GSM4686103 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686103 GSM4686103 GEO Accession GSM4686103 SRX8808174 GSM4686104 SRP273093 PRJNA647773 SRS7074235 GSM4686104 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686104 GSM4686104 GEO Accession GSM4686104 SRX8808175 GSM4686105 SRP273093 PRJNA647773 SRS7074236 GSM4686105 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686105 GSM4686105 GEO Accession GSM4686105 SRX8808176 GSM4686106 SRP273093 PRJNA647773 SRS7074237 GSM4686106 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686106 GSM4686106 GEO Accession GSM4686106 SRX8808177 GSM4686107 SRP273093 PRJNA647773 SRS7074238 GSM4686107 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686107 GSM4686107 GEO Accession GSM4686107 SRX8808178 GSM4686108 SRP273093 PRJNA647773 SRS7074239 GSM4686108 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686108 GSM4686108 GEO Accession GSM4686108 SRX8808179 GSM4686109 SRP273093 PRJNA647773 SRS7074240 GSM4686109 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686109 GSM4686109 GEO Accession GSM4686109 SRX8808180 GSM4686110 SRP273093 PRJNA647773 SRS7074241 GSM4686110 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686110 GSM4686110 GEO Accession GSM4686110 SRX8808181 GSM4686111 SRP273093 PRJNA647773 SRS7074242 GSM4686111 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686111 GSM4686111 GEO Accession GSM4686111 SRX8808182 GSM4686112 SRP273093 PRJNA647773 SRS7074243 GSM4686112 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686112 GSM4686112 GEO Accession GSM4686112 SRX8808183 GSM4686113 SRP273093 PRJNA647773 SRS7074244 GSM4686113 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686113 GSM4686113 GEO Accession GSM4686113 SRX8808184 GSM4686114 SRP273093 PRJNA647773 SRS7074245 GSM4686114 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686114 GSM4686114 GEO Accession GSM4686114 SRX8808185 GSM4686115 SRP273093 PRJNA647773 SRS7074246 GSM4686115 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686115 GSM4686115 GEO Accession GSM4686115 SRX8808186 GSM4686116 SRP273093 PRJNA647773 SRS7074247 GSM4686116 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686116 GSM4686116 GEO Accession GSM4686116 SRX8808187 GSM4686117 SRP273093 PRJNA647773 SRS7074248 GSM4686117 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686117 GSM4686117 GEO Accession GSM4686117 SRX8808188 GSM4686118 SRP273093 PRJNA647773 SRS7074249 GSM4686118 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686118 GSM4686118 GEO Accession GSM4686118 SRX8808189 GSM4686119 SRP273093 PRJNA647773 SRS7074250 GSM4686119 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686119 GSM4686119 GEO Accession GSM4686119 SRX8808190 GSM4686120 SRP273093 PRJNA647773 SRS7074251 GSM4686120 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686120 GSM4686120 GEO Accession GSM4686120 SRX8808191 GSM4686121 SRP273093 PRJNA647773 SRS7074252 GSM4686121 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686121 GSM4686121 GEO Accession GSM4686121 SRX8808192 GSM4686122 SRP273093 PRJNA647773 SRS7074253 GSM4686122 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686122 GSM4686122 GEO Accession GSM4686122 SRX8808193 GSM4686123 SRP273093 PRJNA647773 SRS7074254 GSM4686123 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686123 GSM4686123 GEO Accession GSM4686123 SRX8808194 GSM4686124 SRP273093 PRJNA647773 SRS7074255 GSM4686124 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686124 GSM4686124 GEO Accession GSM4686124 SRX8808195 GSM4686125 SRP273093 PRJNA647773 SRS7074256 GSM4686125 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686125 GSM4686125 GEO Accession GSM4686125 SRX8808196 GSM4686126 SRP273093 PRJNA647773 SRS7074257 GSM4686126 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686126 GSM4686126 GEO Accession GSM4686126 SRX8808197 GSM4686127 SRP273093 PRJNA647773 SRS7074258 GSM4686127 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686127 GSM4686127 GEO Accession GSM4686127 SRX8808198 GSM4686128 SRP273093 PRJNA647773 SRS7074259 GSM4686128 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686128 GSM4686128 GEO Accession GSM4686128 SRX8808199 GSM4686129 SRP273093 PRJNA647773 SRS7074260 GSM4686129 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686129 GSM4686129 GEO Accession GSM4686129 SRX8808200 GSM4686130 SRP273093 PRJNA647773 SRS7074261 GSM4686130 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686130 GSM4686130 GEO Accession GSM4686130 SRX8808201 GSM4686131 SRP273093 PRJNA647773 SRS7074262 GSM4686131 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686131 GSM4686131 GEO Accession GSM4686131 SRX8808202 GSM4686132 SRP273093 PRJNA647773 SRS7074263 GSM4686132 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686132 GSM4686132 GEO Accession GSM4686132 SRX8808203 GSM4686133 SRP273093 PRJNA647773 SRS7074264 GSM4686133 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686133 GSM4686133 GEO Accession GSM4686133 SRX8808204 GSM4686134 SRP273093 PRJNA647773 SRS7074265 GSM4686134 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686134 GSM4686134 GEO Accession GSM4686134 SRX8808205 GSM4686135 SRP273093 PRJNA647773 SRS7074266 GSM4686135 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686135 GSM4686135 GEO Accession GSM4686135 SRX8808206 GSM4686136 SRP273093 PRJNA647773 SRS7074267 GSM4686136 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686136 GSM4686136 GEO Accession GSM4686136 SRX8808207 GSM4686137 SRP273093 PRJNA647773 SRS7074268 GSM4686137 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686137 GSM4686137 GEO Accession GSM4686137 SRX8808208 GSM4686138 SRP273093 PRJNA647773 SRS7074269 GSM4686138 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686138 GSM4686138 GEO Accession GSM4686138 SRX8808209 GSM4686139 SRP273093 PRJNA647773 SRS7074270 GSM4686139 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686139 GSM4686139 GEO Accession GSM4686139 SRX8808210 GSM4686140 SRP273093 PRJNA647773 SRS7074274 GSM4686140 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686140 GSM4686140 GEO Accession GSM4686140 SRX8808211 GSM4686141 SRP273093 PRJNA647773 SRS7074271 GSM4686141 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686141 GSM4686141 GEO Accession GSM4686141 SRX8808212 GSM4686142 SRP273093 PRJNA647773 SRS7074272 GSM4686142 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686142 GSM4686142 GEO Accession GSM4686142 SRX8808213 GSM4686143 SRP273093 PRJNA647773 SRS7074273 GSM4686143 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686143 GSM4686143 GEO Accession GSM4686143 SRX8808214 GSM4686144 SRP273093 PRJNA647773 SRS7074276 GSM4686144 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686144 GSM4686144 GEO Accession GSM4686144 SRX8808215 GSM4686145 SRP273093 PRJNA647773 SRS7074275 GSM4686145 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686145 GSM4686145 GEO Accession GSM4686145 SRX8808216 GSM4686146 SRP273093 PRJNA647773 SRS7074277 GSM4686146 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686146 GSM4686146 GEO Accession GSM4686146 SRX8808217 GSM4686147 SRP273093 PRJNA647773 SRS7074279 GSM4686147 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686147 GSM4686147 GEO Accession GSM4686147 SRX8808218 GSM4686148 SRP273093 PRJNA647773 SRS7074278 GSM4686148 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686148 GSM4686148 GEO Accession GSM4686148 SRX8808219 GSM4686149 SRP273093 PRJNA647773 SRS7074280 GSM4686149 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686149 GSM4686149 GEO Accession GSM4686149 SRX8808220 GSM4686150 SRP273093 PRJNA647773 SRS7074281 GSM4686150 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686150 GSM4686150 GEO Accession GSM4686150 SRX8808221 GSM4686151 SRP273093 PRJNA647773 SRS7074282 GSM4686151 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686151 GSM4686151 GEO Accession GSM4686151 SRX8808222 GSM4686152 SRP273093 PRJNA647773 SRS7074283 GSM4686152 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686152 GSM4686152 GEO Accession GSM4686152 SRX8808223 GSM4686153 SRP273093 PRJNA647773 SRS7074284 GSM4686153 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686153 GSM4686153 GEO Accession GSM4686153 SRX8808224 GSM4686154 SRP273093 PRJNA647773 SRS7074286 GSM4686154 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686154 GSM4686154 GEO Accession GSM4686154 SRX8808225 GSM4686155 SRP273093 PRJNA647773 SRS7074285 GSM4686155 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686155 GSM4686155 GEO Accession GSM4686155 SRX8808226 GSM4686156 SRP273093 PRJNA647773 SRS7074287 GSM4686156 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686156 GSM4686156 GEO Accession GSM4686156 SRX8808227 GSM4686157 SRP273093 PRJNA647773 SRS7074288 GSM4686157 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686157 GSM4686157 GEO Accession GSM4686157 SRX8808228 GSM4686158 SRP273093 PRJNA647773 SRS7074289 GSM4686158 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686158 GSM4686158 GEO Accession GSM4686158 SRX8808229 GSM4686159 SRP273093 PRJNA647773 SRS7074290 GSM4686159 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686159 GSM4686159 GEO Accession GSM4686159 SRX8808230 GSM4686160 SRP273093 PRJNA647773 SRS7074291 GSM4686160 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686160 GSM4686160 GEO Accession GSM4686160 SRX8808231 GSM4686161 SRP273093 PRJNA647773 SRS7074293 GSM4686161 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686161 GSM4686161 GEO Accession GSM4686161 SRX8808232 GSM4686162 SRP273093 PRJNA647773 SRS7074292 GSM4686162 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686162 GSM4686162 GEO Accession GSM4686162 SRX8808233 GSM4686163 SRP273093 PRJNA647773 SRS7074294 GSM4686163 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686163 GSM4686163 GEO Accession GSM4686163 SRX8808234 GSM4686164 SRP273093 PRJNA647773 SRS7074295 GSM4686164 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686164 GSM4686164 GEO Accession GSM4686164 SRX8808235 GSM4686165 SRP273093 PRJNA647773 SRS7074296 GSM4686165 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686165 GSM4686165 GEO Accession GSM4686165 SRX8808236 GSM4686166 SRP273093 PRJNA647773 SRS7074297 GSM4686166 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686166 GSM4686166 GEO Accession GSM4686166 SRX8808237 GSM4686167 SRP273093 PRJNA647773 SRS7074298 GSM4686167 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686167 GSM4686167 GEO Accession GSM4686167 SRX8808238 GSM4686168 SRP273093 PRJNA647773 SRS7074300 GSM4686168 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686168 GSM4686168 GEO Accession GSM4686168 SRX8808239 GSM4686169 SRP273093 PRJNA647773 SRS7074299 GSM4686169 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686169 GSM4686169 GEO Accession GSM4686169 SRX8808240 GSM4686170 SRP273093 PRJNA647773 SRS7074301 GSM4686170 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686170 GSM4686170 GEO Accession GSM4686170 SRX8808241 GSM4686171 SRP273093 PRJNA647773 SRS7074302 GSM4686171 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686171 GSM4686171 GEO Accession GSM4686171 SRX8808242 GSM4686172 SRP273093 PRJNA647773 SRS7074303 GSM4686172 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686172 GSM4686172 GEO Accession GSM4686172 SRX8808243 GSM4686173 SRP273093 PRJNA647773 SRS7074304 GSM4686173 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686173 GSM4686173 GEO Accession GSM4686173 SRX8808244 GSM4686174 SRP273093 PRJNA647773 SRS7074305 GSM4686174 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686174 GSM4686174 GEO Accession GSM4686174 SRX8808245 GSM4686175 SRP273093 PRJNA647773 SRS7074306 GSM4686175 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686175 GSM4686175 GEO Accession GSM4686175 SRX8808246 GSM4686176 SRP273093 PRJNA647773 SRS7074307 GSM4686176 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686176 GSM4686176 GEO Accession GSM4686176 SRX8808247 GSM4686177 SRP273093 PRJNA647773 SRS7074308 GSM4686177 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686177 GSM4686177 GEO Accession GSM4686177 SRX8808248 GSM4686178 SRP273093 PRJNA647773 SRS7074309 GSM4686178 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686178 GSM4686178 GEO Accession GSM4686178 SRX8808249 GSM4686179 SRP273093 PRJNA647773 SRS7074310 GSM4686179 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686179 GSM4686179 GEO Accession GSM4686179 SRX8808250 GSM4686180 SRP273093 PRJNA647773 SRS7074311 GSM4686180 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686180 GSM4686180 GEO Accession GSM4686180 SRX8808251 GSM4686181 SRP273093 PRJNA647773 SRS7074312 GSM4686181 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686181 GSM4686181 GEO Accession GSM4686181 SRX8808252 GSM4686182 SRP273093 PRJNA647773 SRS7074315 GSM4686182 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686182 GSM4686182 GEO Accession GSM4686182 SRX8808253 GSM4686183 SRP273093 PRJNA647773 SRS7074313 GSM4686183 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686183 GSM4686183 GEO Accession GSM4686183 SRX8808254 GSM4686184 SRP273093 PRJNA647773 SRS7074314 GSM4686184 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686184 GSM4686184 GEO Accession GSM4686184 SRX8808255 GSM4686185 SRP273093 PRJNA647773 SRS7074316 GSM4686185 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686185 GSM4686185 GEO Accession GSM4686185 SRX8808256 GSM4686186 SRP273093 PRJNA647773 SRS7074319 GSM4686186 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686186 GSM4686186 GEO Accession GSM4686186 SRX8808257 GSM4686187 SRP273093 PRJNA647773 SRS7074318 GSM4686187 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686187 GSM4686187 GEO Accession GSM4686187 SRX8808258 GSM4686188 SRP273093 PRJNA647773 SRS7074317 GSM4686188 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686188 GSM4686188 GEO Accession GSM4686188 SRX8808259 GSM4686189 SRP273093 PRJNA647773 SRS7074322 GSM4686189 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686189 GSM4686189 GEO Accession GSM4686189 SRX8808260 GSM4686190 SRP273093 PRJNA647773 SRS7074320 GSM4686190 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686190 GSM4686190 GEO Accession GSM4686190 SRX8808261 GSM4686191 SRP273093 PRJNA647773 SRS7074321 GSM4686191 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686191 GSM4686191 GEO Accession GSM4686191 SRX8808262 GSM4686192 SRP273093 PRJNA647773 SRS7074323 GSM4686192 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686192 GSM4686192 GEO Accession GSM4686192 SRX8808263 GSM4686193 SRP273093 PRJNA647773 SRS7074324 GSM4686193 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686193 GSM4686193 GEO Accession GSM4686193 SRX8808264 GSM4686194 SRP273093 PRJNA647773 SRS7074325 GSM4686194 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686194 GSM4686194 GEO Accession GSM4686194 SRX8808265 GSM4686195 SRP273093 PRJNA647773 SRS7074326 GSM4686195 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686195 GSM4686195 GEO Accession GSM4686195 SRX8808266 GSM4686196 SRP273093 PRJNA647773 SRS7074328 GSM4686196 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686196 GSM4686196 GEO Accession GSM4686196 SRX8808267 GSM4686197 SRP273093 PRJNA647773 SRS7074327 GSM4686197 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686197 GSM4686197 GEO Accession GSM4686197 SRX8808268 GSM4686198 SRP273093 PRJNA647773 SRS7074329 GSM4686198 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686198 GSM4686198 GEO Accession GSM4686198 SRX8808269 GSM4686199 SRP273093 PRJNA647773 SRS7074330 GSM4686199 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686199 GSM4686199 GEO Accession GSM4686199 SRX8808270 GSM4686200 SRP273093 PRJNA647773 SRS7074331 GSM4686200 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686200 GSM4686200 GEO Accession GSM4686200 SRX8808271 GSM4686201 SRP273093 PRJNA647773 SRS7074332 GSM4686201 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686201 GSM4686201 GEO Accession GSM4686201 SRX8808272 GSM4686202 SRP273093 PRJNA647773 SRS7074333 GSM4686202 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686202 GSM4686202 GEO Accession GSM4686202 SRX8808273 GSM4686203 SRP273093 PRJNA647773 SRS7074335 GSM4686203 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686203 GSM4686203 GEO Accession GSM4686203 SRX8808274 GSM4686204 SRP273093 PRJNA647773 SRS7074334 GSM4686204 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686204 GSM4686204 GEO Accession GSM4686204 SRX8808275 GSM4686205 SRP273093 PRJNA647773 SRS7074336 GSM4686205 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686205 GSM4686205 GEO Accession GSM4686205 SRX8808276 GSM4686206 SRP273093 PRJNA647773 SRS7074337 GSM4686206 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686206 GSM4686206 GEO Accession GSM4686206 SRX8808277 GSM4686207 SRP273093 PRJNA647773 SRS7074338 GSM4686207 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686207 GSM4686207 GEO Accession GSM4686207 SRX8808278 GSM4686208 SRP273093 PRJNA647773 SRS7074339 GSM4686208 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686208 GSM4686208 GEO Accession GSM4686208 SRX8808279 GSM4686209 SRP273093 PRJNA647773 SRS7074340 GSM4686209 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686209 GSM4686209 GEO Accession GSM4686209 SRX8808280 GSM4686210 SRP273093 PRJNA647773 SRS7074342 GSM4686210 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686210 GSM4686210 GEO Accession GSM4686210 SRX8808281 GSM4686211 SRP273093 PRJNA647773 SRS7074341 GSM4686211 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686211 GSM4686211 GEO Accession GSM4686211 SRX8808282 GSM4686212 SRP273093 PRJNA647773 SRS7074343 GSM4686212 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686212 GSM4686212 GEO Accession GSM4686212 SRX8808283 GSM4686213 SRP273093 PRJNA647773 SRS7074344 GSM4686213 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686213 GSM4686213 GEO Accession GSM4686213 SRX8808284 GSM4686214 SRP273093 PRJNA647773 SRS7074345 GSM4686214 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686214 GSM4686214 GEO Accession GSM4686214 SRX8808285 GSM4686215 SRP273093 PRJNA647773 SRS7074346 GSM4686215 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686215 GSM4686215 GEO Accession GSM4686215 SRX8808286 GSM4686216 SRP273093 PRJNA647773 SRS7074347 GSM4686216 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686216 GSM4686216 GEO Accession GSM4686216 SRX8808287 GSM4686217 SRP273093 PRJNA647773 SRS7074348 GSM4686217 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686217 GSM4686217 GEO Accession GSM4686217 SRX8808288 GSM4686218 SRP273093 PRJNA647773 SRS7074349 GSM4686218 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686218 GSM4686218 GEO Accession GSM4686218 SRX8808289 GSM4686219 SRP273093 PRJNA647773 SRS7074350 GSM4686219 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686219 GSM4686219 GEO Accession GSM4686219 SRX8808290 GSM4686220 SRP273093 PRJNA647773 SRS7074351 GSM4686220 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686220 GSM4686220 GEO Accession GSM4686220 SRX8808291 GSM4686221 SRP273093 PRJNA647773 SRS7074352 GSM4686221 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686221 GSM4686221 GEO Accession GSM4686221 SRX8808292 GSM4686222 SRP273093 PRJNA647773 SRS7074353 GSM4686222 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686222 GSM4686222 GEO Accession GSM4686222 SRX8808293 GSM4686223 SRP273093 PRJNA647773 SRS7074354 GSM4686223 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686223 GSM4686223 GEO Accession GSM4686223 SRX8808294 GSM4686224 SRP273093 PRJNA647773 SRS7074355 GSM4686224 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686224 GSM4686224 GEO Accession GSM4686224 SRX8808295 GSM4686225 SRP273093 PRJNA647773 SRS7074356 GSM4686225 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686225 GSM4686225 GEO Accession GSM4686225 SRX8808296 GSM4686226 SRP273093 PRJNA647773 SRS7074357 GSM4686226 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686226 GSM4686226 GEO Accession GSM4686226 SRX8808297 GSM4686227 SRP273093 PRJNA647773 SRS7074358 GSM4686227 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686227 GSM4686227 GEO Accession GSM4686227 SRX8808298 GSM4686228 SRP273093 PRJNA647773 SRS7074359 GSM4686228 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686228 GSM4686228 GEO Accession GSM4686228 SRX8808299 GSM4686229 SRP273093 PRJNA647773 SRS7074360 GSM4686229 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686229 GSM4686229 GEO Accession GSM4686229 SRX8808300 GSM4686230 SRP273093 PRJNA647773 SRS7074361 GSM4686230 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686230 GSM4686230 GEO Accession GSM4686230 SRX8808301 GSM4686231 SRP273093 PRJNA647773 SRS7074363 GSM4686231 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686231 GSM4686231 GEO Accession GSM4686231 SRX8808302 GSM4686232 SRP273093 PRJNA647773 SRS7074362 GSM4686232 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686232 GSM4686232 GEO Accession GSM4686232 SRX8808303 GSM4686233 SRP273093 PRJNA647773 SRS7074366 GSM4686233 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686233 GSM4686233 GEO Accession GSM4686233 SRX8808304 GSM4686234 SRP273093 PRJNA647773 SRS7074364 GSM4686234 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686234 GSM4686234 GEO Accession GSM4686234 SRX8808305 GSM4686235 SRP273093 PRJNA647773 SRS7074365 GSM4686235 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686235 GSM4686235 GEO Accession GSM4686235 SRX8808306 GSM4686236 SRP273093 PRJNA647773 SRS7074367 GSM4686236 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686236 GSM4686236 GEO Accession GSM4686236 SRX8808307 GSM4686237 SRP273093 PRJNA647773 SRS7074369 GSM4686237 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686237 GSM4686237 GEO Accession GSM4686237 SRX8808308 GSM4686238 SRP273093 PRJNA647773 SRS7074368 GSM4686238 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686238 GSM4686238 GEO Accession GSM4686238 SRX8808309 GSM4686239 SRP273093 PRJNA647773 SRS7074372 GSM4686239 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686239 GSM4686239 GEO Accession GSM4686239 SRX8808310 GSM4686240 SRP273093 PRJNA647773 SRS7074371 GSM4686240 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686240 GSM4686240 GEO Accession GSM4686240 SRX8808311 GSM4686241 SRP273093 PRJNA647773 SRS7074370 GSM4686241 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686241 GSM4686241 GEO Accession GSM4686241 SRX8808312 GSM4686242 SRP273093 PRJNA647773 SRS7074373 GSM4686242 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686242 GSM4686242 GEO Accession GSM4686242 SRX8808313 GSM4686243 SRP273093 PRJNA647773 SRS7074374 GSM4686243 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686243 GSM4686243 GEO Accession GSM4686243 SRX8808314 GSM4686244 SRP273093 PRJNA647773 SRS7074375 GSM4686244 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686244 GSM4686244 GEO Accession GSM4686244 SRX8808315 GSM4686245 SRP273093 PRJNA647773 SRS7074376 GSM4686245 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686245 GSM4686245 GEO Accession GSM4686245 SRX8808316 GSM4686246 SRP273093 PRJNA647773 SRS7074379 GSM4686246 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686246 GSM4686246 GEO Accession GSM4686246 SRX8808317 GSM4686247 SRP273093 PRJNA647773 SRS7074377 GSM4686247 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686247 GSM4686247 GEO Accession GSM4686247 SRX8808318 GSM4686248 SRP273093 PRJNA647773 SRS7074378 GSM4686248 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686248 GSM4686248 GEO Accession GSM4686248 SRX8808319 GSM4686249 SRP273093 PRJNA647773 SRS7074380 GSM4686249 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686249 GSM4686249 GEO Accession GSM4686249 SRX8808320 GSM4686250 SRP273093 PRJNA647773 SRS7074381 GSM4686250 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686250 GSM4686250 GEO Accession GSM4686250 SRX8808321 GSM4686251 SRP273093 PRJNA647773 SRS7074382 GSM4686251 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686251 GSM4686251 GEO Accession GSM4686251 SRX8808322 GSM4686252 SRP273093 PRJNA647773 SRS7074383 GSM4686252 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686252 GSM4686252 GEO Accession GSM4686252 SRX8808323 GSM4686253 SRP273093 PRJNA647773 SRS7074384 GSM4686253 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686253 GSM4686253 GEO Accession GSM4686253 SRX8808324 GSM4686254 SRP273093 PRJNA647773 SRS7074385 GSM4686254 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686254 GSM4686254 GEO Accession GSM4686254 SRX8808325 GSM4686255 SRP273093 PRJNA647773 SRS7074386 GSM4686255 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686255 GSM4686255 GEO Accession GSM4686255 SRX8808326 GSM4686256 SRP273093 PRJNA647773 SRS7074387 GSM4686256 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686256 GSM4686256 GEO Accession GSM4686256 SRX8808327 GSM4686257 SRP273093 PRJNA647773 SRS7074388 GSM4686257 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686257 GSM4686257 GEO Accession GSM4686257 SRX8808328 GSM4686258 SRP273093 PRJNA647773 SRS7074389 GSM4686258 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686258 GSM4686258 GEO Accession GSM4686258 SRX8808329 GSM4686259 SRP273093 PRJNA647773 SRS7074390 GSM4686259 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686259 GSM4686259 GEO Accession GSM4686259 SRX8808330 GSM4686260 SRP273093 PRJNA647773 SRS7074392 GSM4686260 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686260 GSM4686260 GEO Accession GSM4686260 SRX8808331 GSM4686261 SRP273093 PRJNA647773 SRS7074391 GSM4686261 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686261 GSM4686261 GEO Accession GSM4686261 SRX8808332 GSM4686262 SRP273093 PRJNA647773 SRS7074393 GSM4686262 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686262 GSM4686262 GEO Accession GSM4686262 SRX8808333 GSM4686263 SRP273093 PRJNA647773 SRS7074394 GSM4686263 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686263 GSM4686263 GEO Accession GSM4686263 SRX8808334 GSM4686264 SRP273093 PRJNA647773 SRS7074395 GSM4686264 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686264 GSM4686264 GEO Accession GSM4686264 SRX8808335 GSM4686265 SRP273093 PRJNA647773 SRS7074396 GSM4686265 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686265 GSM4686265 GEO Accession GSM4686265 SRX8808336 GSM4686266 SRP273093 PRJNA647773 SRS7074397 GSM4686266 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686266 GSM4686266 GEO Accession GSM4686266 SRX8808337 GSM4686267 SRP273093 PRJNA647773 SRS7074398 GSM4686267 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686267 GSM4686267 GEO Accession GSM4686267 SRX8808338 GSM4686268 SRP273093 PRJNA647773 SRS7074399 GSM4686268 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686268 GSM4686268 GEO Accession GSM4686268 SRX8808339 GSM4686269 SRP273093 PRJNA647773 SRS7074400 GSM4686269 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686269 GSM4686269 GEO Accession GSM4686269 SRX8808340 GSM4686270 SRP273093 PRJNA647773 SRS7074402 GSM4686270 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686270 GSM4686270 GEO Accession GSM4686270 SRX8808341 GSM4686271 SRP273093 PRJNA647773 SRS7074401 GSM4686271 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686271 GSM4686271 GEO Accession GSM4686271 SRX8808342 GSM4686272 SRP273093 PRJNA647773 SRS7074403 GSM4686272 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686272 GSM4686272 GEO Accession GSM4686272 SRX8808343 GSM4686273 SRP273093 PRJNA647773 SRS7074408 GSM4686273 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686273 GSM4686273 GEO Accession GSM4686273 SRX8808344 GSM4686274 SRP273093 PRJNA647773 SRS7074405 GSM4686274 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686274 GSM4686274 GEO Accession GSM4686274 SRX8808345 GSM4686275 SRP273093 PRJNA647773 SRS7074404 GSM4686275 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686275 GSM4686275 GEO Accession GSM4686275 SRX8808346 GSM4686276 SRP273093 PRJNA647773 SRS7074406 GSM4686276 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686276 GSM4686276 GEO Accession GSM4686276 SRX8808347 GSM4686277 SRP273093 PRJNA647773 SRS7074411 GSM4686277 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686277 GSM4686277 GEO Accession GSM4686277 SRX8808348 GSM4686278 SRP273093 PRJNA647773 SRS7074407 GSM4686278 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686278 GSM4686278 GEO Accession GSM4686278 SRX8808349 GSM4686279 SRP273093 PRJNA647773 SRS7074409 GSM4686279 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686279 GSM4686279 GEO Accession GSM4686279 SRX8808350 GSM4686280 SRP273093 PRJNA647773 SRS7074413 GSM4686280 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686280 GSM4686280 GEO Accession GSM4686280 SRX8808351 GSM4686281 SRP273093 PRJNA647773 SRS7074410 GSM4686281 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686281 GSM4686281 GEO Accession GSM4686281 SRX8808352 GSM4686282 SRP273093 PRJNA647773 SRS7074414 GSM4686282 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686282 GSM4686282 GEO Accession GSM4686282 SRX8808353 GSM4686283 SRP273093 PRJNA647773 SRS7074412 GSM4686283 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686283 GSM4686283 GEO Accession GSM4686283 SRX8808354 GSM4686284 SRP273093 PRJNA647773 SRS7074415 GSM4686284 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686284 GSM4686284 GEO Accession GSM4686284 SRX8808355 GSM4686285 SRP273093 PRJNA647773 SRS7074417 GSM4686285 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686285 GSM4686285 GEO Accession GSM4686285 SRX8808356 GSM4686286 SRP273093 PRJNA647773 SRS7074416 GSM4686286 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686286 GSM4686286 GEO Accession GSM4686286 SRX8808357 GSM4686287 SRP273093 PRJNA647773 SRS7074421 GSM4686287 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686287 GSM4686287 GEO Accession GSM4686287 SRX8808358 GSM4686288 SRP273093 PRJNA647773 SRS7074418 GSM4686288 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686288 GSM4686288 GEO Accession GSM4686288 SRX8808359 GSM4686289 SRP273093 PRJNA647773 SRS7074419 GSM4686289 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686289 GSM4686289 GEO Accession GSM4686289 SRX8808360 GSM4686290 SRP273093 PRJNA647773 SRS7074420 GSM4686290 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686290 GSM4686290 GEO Accession GSM4686290 SRX8808361 GSM4686291 SRP273093 PRJNA647773 SRS7074422 GSM4686291 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686291 GSM4686291 GEO Accession GSM4686291 SRX8808362 GSM4686292 SRP273093 PRJNA647773 SRS7074423 GSM4686292 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686292 GSM4686292 GEO Accession GSM4686292 SRX8808363 GSM4686293 SRP273093 PRJNA647773 SRS7074424 GSM4686293 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686293 GSM4686293 GEO Accession GSM4686293 SRX8808364 GSM4686294 SRP273093 PRJNA647773 SRS7074426 GSM4686294 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686294 GSM4686294 GEO Accession GSM4686294 SRX8808365 GSM4686295 SRP273093 PRJNA647773 SRS7074425 GSM4686295 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686295 GSM4686295 GEO Accession GSM4686295 SRX8808366 GSM4686296 SRP273093 PRJNA647773 SRS7074427 GSM4686296 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686296 GSM4686296 GEO Accession GSM4686296 SRX8808367 GSM4686297 SRP273093 PRJNA647773 SRS7074428 GSM4686297 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686297 GSM4686297 GEO Accession GSM4686297 SRX8808368 GSM4686298 SRP273093 PRJNA647773 SRS7074429 GSM4686298 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686298 GSM4686298 GEO Accession GSM4686298 SRX8808369 GSM4686299 SRP273093 PRJNA647773 SRS7074430 GSM4686299 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686299 GSM4686299 GEO Accession GSM4686299 SRX8808370 GSM4686300 SRP273093 PRJNA647773 SRS7074431 GSM4686300 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686300 GSM4686300 GEO Accession GSM4686300 SRX8808371 GSM4686301 SRP273093 PRJNA647773 SRS7074432 GSM4686301 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686301 GSM4686301 GEO Accession GSM4686301 SRX8808372 GSM4686302 SRP273093 PRJNA647773 SRS7074433 GSM4686302 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686302 GSM4686302 GEO Accession GSM4686302 SRX8808373 GSM4686303 SRP273093 PRJNA647773 SRS7074434 GSM4686303 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686303 GSM4686303 GEO Accession GSM4686303 SRX8808374 GSM4686304 SRP273093 PRJNA647773 SRS7074436 GSM4686304 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686304 GSM4686304 GEO Accession GSM4686304 SRX8808375 GSM4686305 SRP273093 PRJNA647773 SRS7074435 GSM4686305 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686305 GSM4686305 GEO Accession GSM4686305 SRX8808376 GSM4686306 SRP273093 PRJNA647773 SRS7074437 GSM4686306 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686306 GSM4686306 GEO Accession GSM4686306 SRX8808377 GSM4686307 SRP273093 PRJNA647773 SRS7074438 GSM4686307 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686307 GSM4686307 GEO Accession GSM4686307 SRX8808378 GSM4686308 SRP273093 PRJNA647773 SRS7074439 GSM4686308 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686308 GSM4686308 GEO Accession GSM4686308 SRX8808379 GSM4686309 SRP273093 PRJNA647773 SRS7074440 GSM4686309 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686309 GSM4686309 GEO Accession GSM4686309 SRX8808380 GSM4686310 SRP273093 PRJNA647773 SRS7074441 GSM4686310 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686310 GSM4686310 GEO Accession GSM4686310 SRX8808381 GSM4686311 SRP273093 PRJNA647773 SRS7074442 GSM4686311 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686311 GSM4686311 GEO Accession GSM4686311 SRX8808382 GSM4686312 SRP273093 PRJNA647773 SRS7074443 GSM4686312 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686312 GSM4686312 GEO Accession GSM4686312 SRX8808383 GSM4686313 SRP273093 PRJNA647773 SRS7074444 GSM4686313 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686313 GSM4686313 GEO Accession GSM4686313 SRX8808384 GSM4686314 SRP273093 PRJNA647773 SRS7074445 GSM4686314 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686314 GSM4686314 GEO Accession GSM4686314 SRX8808385 GSM4686315 SRP273093 PRJNA647773 SRS7074446 GSM4686315 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686315 GSM4686315 GEO Accession GSM4686315 SRX8808386 GSM4686316 SRP273093 PRJNA647773 SRS7074447 GSM4686316 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686316 GSM4686316 GEO Accession GSM4686316 SRX8808387 GSM4686317 SRP273093 PRJNA647773 SRS7074448 GSM4686317 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686317 GSM4686317 GEO Accession GSM4686317 SRX8808388 GSM4686318 SRP273093 PRJNA647773 SRS7074449 GSM4686318 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686318 GSM4686318 GEO Accession GSM4686318 SRX8808389 GSM4686319 SRP273093 PRJNA647773 SRS7074450 GSM4686319 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686319 GSM4686319 GEO Accession GSM4686319 SRX8808390 GSM4686320 SRP273093 PRJNA647773 SRS7074452 GSM4686320 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686320 GSM4686320 GEO Accession GSM4686320 SRX8808391 GSM4686321 SRP273093 PRJNA647773 SRS7074451 GSM4686321 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686321 GSM4686321 GEO Accession GSM4686321 SRX8808392 GSM4686322 SRP273093 PRJNA647773 SRS7074453 GSM4686322 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686322 GSM4686322 GEO Accession GSM4686322 SRX8808393 GSM4686323 SRP273093 PRJNA647773 SRS7074454 GSM4686323 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686323 GSM4686323 GEO Accession GSM4686323 SRX8808394 GSM4686324 SRP273093 PRJNA647773 SRS7074455 GSM4686324 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686324 GSM4686324 GEO Accession GSM4686324 SRX8808395 GSM4686325 SRP273093 PRJNA647773 SRS7074456 GSM4686325 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686325 GSM4686325 GEO Accession GSM4686325 SRX8808396 GSM4686326 SRP273093 PRJNA647773 SRS7074457 GSM4686326 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686326 GSM4686326 GEO Accession GSM4686326 SRX8808397 GSM4686327 SRP273093 PRJNA647773 SRS7074458 GSM4686327 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686327 GSM4686327 GEO Accession GSM4686327 SRX8808398 GSM4686328 SRP273093 PRJNA647773 SRS7074459 GSM4686328 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686328 GSM4686328 GEO Accession GSM4686328 SRX8808399 GSM4686329 SRP273093 PRJNA647773 SRS7074461 GSM4686329 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686329 GSM4686329 GEO Accession GSM4686329 SRX8808400 GSM4686330 SRP273093 PRJNA647773 SRS7074460 GSM4686330 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686330 GSM4686330 GEO Accession GSM4686330 SRX8808401 GSM4686331 SRP273093 PRJNA647773 SRS7074462 GSM4686331 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686331 GSM4686331 GEO Accession GSM4686331 SRX8808402 GSM4686332 SRP273093 PRJNA647773 SRS7074463 GSM4686332 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686332 GSM4686332 GEO Accession GSM4686332 SRX8808403 GSM4686333 SRP273093 PRJNA647773 SRS7074464 GSM4686333 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686333 GSM4686333 GEO Accession GSM4686333 SRX8808404 GSM4686334 SRP273093 PRJNA647773 SRS7074465 GSM4686334 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686334 GSM4686334 GEO Accession GSM4686334 SRX8808405 GSM4686335 SRP273093 PRJNA647773 SRS7074466 GSM4686335 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686335 GSM4686335 GEO Accession GSM4686335 SRX8808406 GSM4686336 SRP273093 PRJNA647773 SRS7074467 GSM4686336 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686336 GSM4686336 GEO Accession GSM4686336 SRX8808407 GSM4686337 SRP273093 PRJNA647773 SRS7074468 GSM4686337 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686337 GSM4686337 GEO Accession GSM4686337 SRX8808408 GSM4686338 SRP273093 PRJNA647773 SRS7074469 GSM4686338 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686338 GSM4686338 GEO Accession GSM4686338 SRX8808409 GSM4686339 SRP273093 PRJNA647773 SRS7074470 GSM4686339 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686339 GSM4686339 GEO Accession GSM4686339 SRX8808410 GSM4686340 SRP273093 PRJNA647773 SRS7074471 GSM4686340 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686340 GSM4686340 GEO Accession GSM4686340 SRX8808411 GSM4686341 SRP273093 PRJNA647773 SRS7074472 GSM4686341 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686341 GSM4686341 GEO Accession GSM4686341 SRX8808412 GSM4686342 SRP273093 PRJNA647773 SRS7074473 GSM4686342 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686342 GSM4686342 GEO Accession GSM4686342 SRX8808413 GSM4686343 SRP273093 PRJNA647773 SRS7074474 GSM4686343 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686343 GSM4686343 GEO Accession GSM4686343 SRX8808414 GSM4686344 SRP273093 PRJNA647773 SRS7074475 GSM4686344 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686344 GSM4686344 GEO Accession GSM4686344 SRX8808415 GSM4686345 SRP273093 PRJNA647773 SRS7074476 GSM4686345 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686345 GSM4686345 GEO Accession GSM4686345 SRX8808416 GSM4686346 SRP273093 PRJNA647773 SRS7074477 GSM4686346 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686346 GSM4686346 GEO Accession GSM4686346 SRX8808417 GSM4686347 SRP273093 PRJNA647773 SRS7074479 GSM4686347 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686347 GSM4686347 GEO Accession GSM4686347 SRX8808418 GSM4686348 SRP273093 PRJNA647773 SRS7074478 GSM4686348 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686348 GSM4686348 GEO Accession GSM4686348 SRX8808419 GSM4686349 SRP273093 PRJNA647773 SRS7074480 GSM4686349 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686349 GSM4686349 GEO Accession GSM4686349 SRX8808420 GSM4686350 SRP273093 PRJNA647773 SRS7074481 GSM4686350 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686350 GSM4686350 GEO Accession GSM4686350 SRX8808421 GSM4686351 SRP273093 PRJNA647773 SRS7074482 GSM4686351 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686351 GSM4686351 GEO Accession GSM4686351 SRX8808422 GSM4686352 SRP273093 PRJNA647773 SRS7074483 GSM4686352 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686352 GSM4686352 GEO Accession GSM4686352 SRX8808423 GSM4686353 SRP273093 PRJNA647773 SRS7074484 GSM4686353 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686353 GSM4686353 GEO Accession GSM4686353 SRX8808424 GSM4686354 SRP273093 PRJNA647773 SRS7074485 GSM4686354 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686354 GSM4686354 GEO Accession GSM4686354 SRX8808425 GSM4686355 SRP273093 PRJNA647773 SRS7074486 GSM4686355 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686355 GSM4686355 GEO Accession GSM4686355 SRX8808426 GSM4686356 SRP273093 PRJNA647773 SRS7074487 GSM4686356 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686356 GSM4686356 GEO Accession GSM4686356 SRX8808427 GSM4686357 SRP273093 PRJNA647773 SRS7074488 GSM4686357 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686357 GSM4686357 GEO Accession GSM4686357 SRX8808428 GSM4686358 SRP273093 PRJNA647773 SRS7074490 GSM4686358 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686358 GSM4686358 GEO Accession GSM4686358 SRX8808429 GSM4686359 SRP273093 PRJNA647773 SRS7074489 GSM4686359 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686359 GSM4686359 GEO Accession GSM4686359 SRX8808430 GSM4686360 SRP273093 PRJNA647773 SRS7074491 GSM4686360 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686360 GSM4686360 GEO Accession GSM4686360 SRX8808431 GSM4686361 SRP273093 PRJNA647773 SRS7074492 GSM4686361 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686361 GSM4686361 GEO Accession GSM4686361 SRX8808432 GSM4686362 SRP273093 PRJNA647773 SRS7074493 GSM4686362 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686362 GSM4686362 GEO Accession GSM4686362 SRX8808433 GSM4686363 SRP273093 PRJNA647773 SRS7074494 GSM4686363 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686363 GSM4686363 GEO Accession GSM4686363 SRX8808434 GSM4686364 SRP273093 PRJNA647773 SRS7074495 GSM4686364 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686364 GSM4686364 GEO Accession GSM4686364 SRX8808435 GSM4686365 SRP273093 PRJNA647773 SRS7074496 GSM4686365 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686365 GSM4686365 GEO Accession GSM4686365 SRX8808436 GSM4686366 SRP273093 PRJNA647773 SRS7074497 GSM4686366 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686366 GSM4686366 GEO Accession GSM4686366 SRX8808437 GSM4686367 SRP273093 PRJNA647773 SRS7074498 GSM4686367 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686367 GSM4686367 GEO Accession GSM4686367 SRX8808438 GSM4686368 SRP273093 PRJNA647773 SRS7074499 GSM4686368 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686368 GSM4686368 GEO Accession GSM4686368 SRX8808439 GSM4686369 SRP273093 PRJNA647773 SRS7074500 GSM4686369 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686369 GSM4686369 GEO Accession GSM4686369 SRX8808440 GSM4686370 SRP273093 PRJNA647773 SRS7074501 GSM4686370 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686370 GSM4686370 GEO Accession GSM4686370 SRX8808441 GSM4686371 SRP273093 PRJNA647773 SRS7074502 GSM4686371 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686371 GSM4686371 GEO Accession GSM4686371 SRX8808443 GSM4686373 SRP273093 PRJNA647773 SRS7074504 GSM4686373 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686373 GSM4686373 GEO Accession GSM4686373 SRX8808444 GSM4686374 SRP273093 PRJNA647773 SRS7074505 GSM4686374 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686374 GSM4686374 GEO Accession GSM4686374 SRX8808445 GSM4686375 SRP273093 PRJNA647773 SRS7074506 GSM4686375 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686375 GSM4686375 GEO Accession GSM4686375 SRX8808446 GSM4686376 SRP273093 PRJNA647773 SRS7074507 GSM4686376 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686376 GSM4686376 GEO Accession GSM4686376 SRX8808447 GSM4686377 SRP273093 PRJNA647773 SRS7074508 GSM4686377 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686377 GSM4686377 GEO Accession GSM4686377 SRX8808448 GSM4686378 SRP273093 PRJNA647773 SRS7074509 GSM4686378 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686378 GSM4686378 GEO Accession GSM4686378 SRX8808449 GSM4686379 SRP273093 PRJNA647773 SRS7074510 GSM4686379 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686379 GSM4686379 GEO Accession GSM4686379 SRX8808450 GSM4686380 SRP273093 PRJNA647773 SRS7074511 GSM4686380 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686380 GSM4686380 GEO Accession GSM4686380 SRX8808451 GSM4686381 SRP273093 PRJNA647773 SRS7074512 GSM4686381 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686381 GSM4686381 GEO Accession GSM4686381 SRX8808452 GSM4686382 SRP273093 PRJNA647773 SRS7074513 GSM4686382 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686382 GSM4686382 GEO Accession GSM4686382 SRX8808453 GSM4686383 SRP273093 PRJNA647773 SRS7074515 GSM4686383 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686383 GSM4686383 GEO Accession GSM4686383 SRX8808455 GSM4686384 SRP273093 PRJNA647773 SRS7074514 GSM4686384 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686384 GSM4686384 GEO Accession GSM4686384 SRX8808456 GSM4686385 SRP273093 PRJNA647773 SRS7074516 GSM4686385 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686385 GSM4686385 GEO Accession GSM4686385 SRX8808457 GSM4686386 SRP273093 PRJNA647773 SRS7074518 GSM4686386 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686386 GSM4686386 GEO Accession GSM4686386 SRX8808458 GSM4686387 SRP273093 PRJNA647773 SRS7074519 GSM4686387 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686387 GSM4686387 GEO Accession GSM4686387 SRX8808459 GSM4686388 SRP273093 PRJNA647773 SRS7074520 GSM4686388 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686388 GSM4686388 GEO Accession GSM4686388 SRX8808460 GSM4686389 SRP273093 PRJNA647773 SRS7074521 GSM4686389 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686389 GSM4686389 GEO Accession GSM4686389 SRX8808461 GSM4686390 SRP273093 PRJNA647773 SRS7074523 GSM4686390 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686390 GSM4686390 GEO Accession GSM4686390 SRX8808462 GSM4686391 SRP273093 PRJNA647773 SRS7074522 GSM4686391 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686391 GSM4686391 GEO Accession GSM4686391 SRX8808463 GSM4686392 SRP273093 PRJNA647773 SRS7074524 GSM4686392 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686392 GSM4686392 GEO Accession GSM4686392 SRX8808464 GSM4686393 SRP273093 PRJNA647773 SRS7074525 GSM4686393 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686393 GSM4686393 GEO Accession GSM4686393 SRX8808465 GSM4686394 SRP273093 PRJNA647773 SRS7074526 GSM4686394 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686394 GSM4686394 GEO Accession GSM4686394 SRX8808466 GSM4686395 SRP273093 PRJNA647773 SRS7074527 GSM4686395 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686395 GSM4686395 GEO Accession GSM4686395 SRX8808467 GSM4686396 SRP273093 PRJNA647773 SRS7074529 GSM4686396 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686396 GSM4686396 GEO Accession GSM4686396 SRX8808468 GSM4686397 SRP273093 PRJNA647773 SRS7074528 GSM4686397 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686397 GSM4686397 GEO Accession GSM4686397 SRX8808469 GSM4686398 SRP273093 PRJNA647773 SRS7074531 GSM4686398 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686398 GSM4686398 GEO Accession GSM4686398 SRX8808470 GSM4686399 SRP273093 PRJNA647773 SRS7074530 GSM4686399 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686399 GSM4686399 GEO Accession GSM4686399 SRX8808471 GSM4686400 SRP273093 PRJNA647773 SRS7074532 GSM4686400 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686400 GSM4686400 GEO Accession GSM4686400 SRX8808472 GSM4686401 SRP273093 PRJNA647773 SRS7074533 GSM4686401 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686401 GSM4686401 GEO Accession GSM4686401 SRX8808473 GSM4686402 SRP273093 PRJNA647773 SRS7074534 GSM4686402 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686402 GSM4686402 GEO Accession GSM4686402 SRX8808474 GSM4686403 SRP273093 PRJNA647773 SRS7074536 GSM4686403 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686403 GSM4686403 GEO Accession GSM4686403 SRX8808475 GSM4686404 SRP273093 PRJNA647773 SRS7074535 GSM4686404 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686404 GSM4686404 GEO Accession GSM4686404 SRX8808476 GSM4686405 SRP273093 PRJNA647773 SRS7074537 GSM4686405 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686405 GSM4686405 GEO Accession GSM4686405 SRX8808477 GSM4686406 SRP273093 PRJNA647773 SRS7074538 GSM4686406 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686406 GSM4686406 GEO Accession GSM4686406 SRX8808478 GSM4686407 SRP273093 PRJNA647773 SRS7074539 GSM4686407 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686407 GSM4686407 GEO Accession GSM4686407 SRX8808479 GSM4686408 SRP273093 PRJNA647773 SRS7074540 GSM4686408 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686408 GSM4686408 GEO Accession GSM4686408 SRX8808480 GSM4686409 SRP273093 PRJNA647773 SRS7074541 GSM4686409 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686409 GSM4686409 GEO Accession GSM4686409 SRX8808481 GSM4686410 SRP273093 PRJNA647773 SRS7074543 GSM4686410 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686410 GSM4686410 GEO Accession GSM4686410 SRX8808482 GSM4686411 SRP273093 PRJNA647773 SRS7074542 GSM4686411 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686411 GSM4686411 GEO Accession GSM4686411 SRX8808483 GSM4686412 SRP273093 PRJNA647773 SRS7074544 GSM4686412 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686412 GSM4686412 GEO Accession GSM4686412 SRX8808484 GSM4686413 SRP273093 PRJNA647773 SRS7074545 GSM4686413 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686413 GSM4686413 GEO Accession GSM4686413 SRX8808485 GSM4686414 SRP273093 PRJNA647773 SRS7074546 GSM4686414 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686414 GSM4686414 GEO Accession GSM4686414 SRX8808486 GSM4686415 SRP273093 PRJNA647773 SRS7074547 GSM4686415 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686415 GSM4686415 GEO Accession GSM4686415 SRX8808487 GSM4686416 SRP273093 PRJNA647773 SRS7074548 GSM4686416 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686416 GSM4686416 GEO Accession GSM4686416 SRX8808488 GSM4686417 SRP273093 PRJNA647773 SRS7074549 GSM4686417 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686417 GSM4686417 GEO Accession GSM4686417 SRX8808489 GSM4686418 SRP273093 PRJNA647773 SRS7074550 GSM4686418 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686418 GSM4686418 GEO Accession GSM4686418 SRX8808491 GSM4686419 SRP273093 PRJNA647773 SRS7074552 GSM4686419 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686419 GSM4686419 GEO Accession GSM4686419 SRX8808492 GSM4686420 SRP273093 PRJNA647773 SRS7074553 GSM4686420 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686420 GSM4686420 GEO Accession GSM4686420 SRX8808493 GSM4686421 SRP273093 PRJNA647773 SRS7074554 GSM4686421 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686421 GSM4686421 GEO Accession GSM4686421 SRX8808494 GSM4686422 SRP273093 PRJNA647773 SRS7074556 GSM4686422 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686422 GSM4686422 GEO Accession GSM4686422 SRX8808495 GSM4686423 SRP273093 PRJNA647773 SRS7074555 GSM4686423 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686423 GSM4686423 GEO Accession GSM4686423 SRX8808496 GSM4686424 SRP273093 PRJNA647773 SRS7074557 GSM4686424 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686424 GSM4686424 GEO Accession GSM4686424 SRX8808497 GSM4686425 SRP273093 PRJNA647773 SRS7074558 GSM4686425 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686425 GSM4686425 GEO Accession GSM4686425 SRX8808498 GSM4686426 SRP273093 PRJNA647773 SRS7074559 GSM4686426 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686426 GSM4686426 GEO Accession GSM4686426 SRX8808499 GSM4686427 SRP273093 PRJNA647773 SRS7074560 GSM4686427 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686427 GSM4686427 GEO Accession GSM4686427 SRX8808500 GSM4686428 SRP273093 PRJNA647773 SRS7074562 GSM4686428 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686428 GSM4686428 GEO Accession GSM4686428 SRX8808501 GSM4686429 SRP273093 PRJNA647773 SRS7074561 GSM4686429 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686429 GSM4686429 GEO Accession GSM4686429 SRX8808502 GSM4686430 SRP273093 PRJNA647773 SRS7074563 GSM4686430 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686430 GSM4686430 GEO Accession GSM4686430 SRX8808503 GSM4686431 SRP273093 PRJNA647773 SRS7074564 GSM4686431 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686431 GSM4686431 GEO Accession GSM4686431 SRX8808504 GSM4686432 SRP273093 PRJNA647773 SRS7074565 GSM4686432 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686432 GSM4686432 GEO Accession GSM4686432 SRX8808505 GSM4686433 SRP273093 PRJNA647773 SRS7074566 GSM4686433 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686433 GSM4686433 GEO Accession GSM4686433 SRX8808506 GSM4686434 SRP273093 PRJNA647773 SRS7074567 GSM4686434 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686434 GSM4686434 GEO Accession GSM4686434 SRX8808507 GSM4686435 SRP273093 PRJNA647773 SRS7074568 GSM4686435 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686435 GSM4686435 GEO Accession GSM4686435 SRX8808508 GSM4686436 SRP273093 PRJNA647773 SRS7074569 GSM4686436 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686436 GSM4686436 GEO Accession GSM4686436 SRX8808509 GSM4686437 SRP273093 PRJNA647773 SRS7074570 GSM4686437 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686437 GSM4686437 GEO Accession GSM4686437 SRX8808510 GSM4686438 SRP273093 PRJNA647773 SRS7074571 GSM4686438 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686438 GSM4686438 GEO Accession GSM4686438 SRX8808511 GSM4686439 SRP273093 PRJNA647773 SRS7074572 GSM4686439 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686439 GSM4686439 GEO Accession GSM4686439 SRX8808512 GSM4686440 SRP273093 PRJNA647773 SRS7074573 GSM4686440 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686440 GSM4686440 GEO Accession GSM4686440 SRX8808513 GSM4686441 SRP273093 PRJNA647773 SRS7074574 GSM4686441 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686441 GSM4686441 GEO Accession GSM4686441 SRX8808514 GSM4686442 SRP273093 PRJNA647773 SRS7074575 GSM4686442 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686442 GSM4686442 GEO Accession GSM4686442 SRX8808515 GSM4686443 SRP273093 PRJNA647773 SRS7074576 GSM4686443 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686443 GSM4686443 GEO Accession GSM4686443 SRX8808516 GSM4686444 SRP273093 PRJNA647773 SRS7074578 GSM4686444 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686444 GSM4686444 GEO Accession GSM4686444 SRX8808517 GSM4686445 SRP273093 PRJNA647773 SRS7074577 GSM4686445 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686445 GSM4686445 GEO Accession GSM4686445 SRX8808518 GSM4686446 SRP273093 PRJNA647773 SRS7074579 GSM4686446 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686446 GSM4686446 GEO Accession GSM4686446 SRX8808519 GSM4686447 SRP273093 PRJNA647773 SRS7074580 GSM4686447 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686447 GSM4686447 GEO Accession GSM4686447 SRX8808520 GSM4686448 SRP273093 PRJNA647773 SRS7074581 GSM4686448 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686448 GSM4686448 GEO Accession GSM4686448 SRX8808521 GSM4686449 SRP273093 PRJNA647773 SRS7074582 GSM4686449 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686449 GSM4686449 GEO Accession GSM4686449 SRX8808522 GSM4686450 SRP273093 PRJNA647773 SRS7074585 GSM4686450 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686450 GSM4686450 GEO Accession GSM4686450 SRX8808523 GSM4686451 SRP273093 PRJNA647773 SRS7074583 GSM4686451 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686451 GSM4686451 GEO Accession GSM4686451 SRX8808524 GSM4686452 SRP273093 PRJNA647773 SRS7074584 GSM4686452 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686452 GSM4686452 GEO Accession GSM4686452 SRX8808525 GSM4686453 SRP273093 PRJNA647773 SRS7074586 GSM4686453 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686453 GSM4686453 GEO Accession GSM4686453 SRX8808526 GSM4686454 SRP273093 PRJNA647773 SRS7074587 GSM4686454 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686454 GSM4686454 GEO Accession GSM4686454 SRX8808527 GSM4686455 SRP273093 PRJNA647773 SRS7074588 GSM4686455 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686455 GSM4686455 GEO Accession GSM4686455 SRX8808528 GSM4686456 SRP273093 PRJNA647773 SRS7074589 GSM4686456 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686456 GSM4686456 GEO Accession GSM4686456 SRX8808529 GSM4686457 SRP273093 PRJNA647773 SRS7074590 GSM4686457 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686457 GSM4686457 GEO Accession GSM4686457 SRX8808530 GSM4686458 SRP273093 PRJNA647773 SRS7074591 GSM4686458 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686458 GSM4686458 GEO Accession GSM4686458 SRX8808531 GSM4686459 SRP273093 PRJNA647773 SRS7074592 GSM4686459 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686459 GSM4686459 GEO Accession GSM4686459 SRX8808532 GSM4686460 SRP273093 PRJNA647773 SRS7074593 GSM4686460 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686460 GSM4686460 GEO Accession GSM4686460 SRX8808534 GSM4686461 SRP273093 PRJNA647773 SRS7074595 GSM4686461 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686461 GSM4686461 GEO Accession GSM4686461 SRX8808535 GSM4686462 SRP273093 PRJNA647773 SRS7074596 GSM4686462 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686462 GSM4686462 GEO Accession GSM4686462 SRX8808536 GSM4686463 SRP273093 PRJNA647773 SRS7074597 GSM4686463 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686463 GSM4686463 GEO Accession GSM4686463 SRX8808537 GSM4686464 SRP273093 PRJNA647773 SRS7074598 GSM4686464 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686464 GSM4686464 GEO Accession GSM4686464 SRX8808538 GSM4686465 SRP273093 PRJNA647773 SRS7074599 GSM4686465 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686465 GSM4686465 GEO Accession GSM4686465 SRX8808539 GSM4686466 SRP273093 PRJNA647773 SRS7074600 GSM4686466 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686466 GSM4686466 GEO Accession GSM4686466 SRX8808540 GSM4686467 SRP273093 PRJNA647773 SRS7074601 GSM4686467 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686467 GSM4686467 GEO Accession GSM4686467 SRX8808541 GSM4686468 SRP273093 PRJNA647773 SRS7074603 GSM4686468 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686468 GSM4686468 GEO Accession GSM4686468 SRX8808542 GSM4686469 SRP273093 PRJNA647773 SRS7074602 GSM4686469 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686469 GSM4686469 GEO Accession GSM4686469 SRX8808543 GSM4686470 SRP273093 PRJNA647773 SRS7074604 GSM4686470 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686470 GSM4686470 GEO Accession GSM4686470 SRX8808544 GSM4686471 SRP273093 PRJNA647773 SRS7074605 GSM4686471 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686471 GSM4686471 GEO Accession GSM4686471 SRX8808545 GSM4686472 SRP273093 PRJNA647773 SRS7074606 GSM4686472 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686472 GSM4686472 GEO Accession GSM4686472 SRX8808546 GSM4686473 SRP273093 PRJNA647773 SRS7074607 GSM4686473 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686473 GSM4686473 GEO Accession GSM4686473 SRX8808547 GSM4686474 SRP273093 PRJNA647773 SRS7074608 GSM4686474 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686474 GSM4686474 GEO Accession GSM4686474 SRX8808548 GSM4686475 SRP273093 PRJNA647773 SRS7074609 GSM4686475 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686475 GSM4686475 GEO Accession GSM4686475 SRX8808549 GSM4686476 SRP273093 PRJNA647773 SRS7075283 GSM4686476 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686476 GSM4686476 GEO Accession GSM4686476 SRX8808550 GSM4686477 SRP273093 PRJNA647773 SRS7075284 GSM4686477 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686477 GSM4686477 GEO Accession GSM4686477 SRX8808551 GSM4686478 SRP273093 PRJNA647773 SRS7075285 GSM4686478 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686478 GSM4686478 GEO Accession GSM4686478 SRX8808552 GSM4686479 SRP273093 PRJNA647773 SRS7075286 GSM4686479 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686479 GSM4686479 GEO Accession GSM4686479 SRX8808553 GSM4686480 SRP273093 PRJNA647773 SRS7075288 GSM4686480 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686480 GSM4686480 GEO Accession GSM4686480 SRX8808554 GSM4686481 SRP273093 PRJNA647773 SRS7075287 GSM4686481 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686481 GSM4686481 GEO Accession GSM4686481 SRX8808555 GSM4686482 SRP273093 PRJNA647773 SRS7075291 GSM4686482 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686482 GSM4686482 GEO Accession GSM4686482 SRX8808556 GSM4686483 SRP273093 PRJNA647773 SRS7075289 GSM4686483 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686483 GSM4686483 GEO Accession GSM4686483 SRX8808557 GSM4686484 SRP273093 PRJNA647773 SRS7075290 GSM4686484 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686484 GSM4686484 GEO Accession GSM4686484 SRX8808558 GSM4686485 SRP273093 PRJNA647773 SRS7075292 GSM4686485 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686485 GSM4686485 GEO Accession GSM4686485 SRX8808559 GSM4686486 SRP273093 PRJNA647773 SRS7075293 GSM4686486 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686486 GSM4686486 GEO Accession GSM4686486 SRX8808560 GSM4686487 SRP273093 PRJNA647773 SRS7075294 GSM4686487 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686487 GSM4686487 GEO Accession GSM4686487 SRX8808561 GSM4686488 SRP273093 PRJNA647773 SRS7074610 GSM4686488 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686488 GSM4686488 GEO Accession GSM4686488 SRX8808562 GSM4686489 SRP273093 PRJNA647773 SRS7074611 GSM4686489 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686489 GSM4686489 GEO Accession GSM4686489 SRX8808563 GSM4686490 SRP273093 PRJNA647773 SRS7074613 GSM4686490 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686490 GSM4686490 GEO Accession GSM4686490 SRX8808564 GSM4686491 SRP273093 PRJNA647773 SRS7074612 GSM4686491 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686491 GSM4686491 GEO Accession GSM4686491 SRX8808565 GSM4686492 SRP273093 PRJNA647773 SRS7074614 GSM4686492 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686492 GSM4686492 GEO Accession GSM4686492 SRX8808566 GSM4686493 SRP273093 PRJNA647773 SRS7074615 GSM4686493 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686493 GSM4686493 GEO Accession GSM4686493 SRX8808567 GSM4686494 SRP273093 PRJNA647773 SRS7075295 GSM4686494 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686494 GSM4686494 GEO Accession GSM4686494 SRX8808568 GSM4686495 SRP273093 PRJNA647773 SRS7075296 GSM4686495 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686495 GSM4686495 GEO Accession GSM4686495 SRX8808569 GSM4686496 SRP273093 PRJNA647773 SRS7074619 GSM4686496 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686496 GSM4686496 GEO Accession GSM4686496 SRX8808570 GSM4686497 SRP273093 PRJNA647773 SRS7074616 GSM4686497 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686497 GSM4686497 GEO Accession GSM4686497 SRX8808571 GSM4686498 SRP273093 PRJNA647773 SRS7074617 GSM4686498 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686498 GSM4686498 GEO Accession GSM4686498 SRX8808572 GSM4686499 SRP273093 PRJNA647773 SRS7074618 GSM4686499 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686499 GSM4686499 GEO Accession GSM4686499 SRX8808573 GSM4686500 SRP273093 PRJNA647773 SRS7074620 GSM4686500 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686500 GSM4686500 GEO Accession GSM4686500 SRX8808574 GSM4686501 SRP273093 PRJNA647773 SRS7074621 GSM4686501 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686501 GSM4686501 GEO Accession GSM4686501 SRX8808575 GSM4686502 SRP273093 PRJNA647773 SRS7074622 GSM4686502 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686502 GSM4686502 GEO Accession GSM4686502 SRX8808576 GSM4686503 SRP273093 PRJNA647773 SRS7074623 GSM4686503 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686503 GSM4686503 GEO Accession GSM4686503 SRX8808577 GSM4686504 SRP273093 PRJNA647773 SRS7074625 GSM4686504 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686504 GSM4686504 GEO Accession GSM4686504 SRX8808578 GSM4686505 SRP273093 PRJNA647773 SRS7074624 GSM4686505 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686505 GSM4686505 GEO Accession GSM4686505 SRX8808579 GSM4686506 SRP273093 PRJNA647773 SRS7074626 GSM4686506 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686506 GSM4686506 GEO Accession GSM4686506 SRX8808580 GSM4686507 SRP273093 PRJNA647773 SRS7074627 GSM4686507 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686507 GSM4686507 GEO Accession GSM4686507 SRX8808581 GSM4686508 SRP273093 PRJNA647773 SRS7074628 GSM4686508 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686508 GSM4686508 GEO Accession GSM4686508 SRX8808582 GSM4686509 SRP273093 PRJNA647773 SRS7074629 GSM4686509 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686509 GSM4686509 GEO Accession GSM4686509 SRX8808583 GSM4686510 SRP273093 PRJNA647773 SRS7074630 GSM4686510 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686510 GSM4686510 GEO Accession GSM4686510 SRX8808584 GSM4686511 SRP273093 PRJNA647773 SRS7074633 GSM4686511 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686511 GSM4686511 GEO Accession GSM4686511 SRX8808585 GSM4686512 SRP273093 PRJNA647773 SRS7074631 GSM4686512 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686512 GSM4686512 GEO Accession GSM4686512 SRX8808586 GSM4686513 SRP273093 PRJNA647773 SRS7074632 GSM4686513 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686513 GSM4686513 GEO Accession GSM4686513 SRX8808587 GSM4686514 SRP273093 PRJNA647773 SRS7074635 GSM4686514 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686514 GSM4686514 GEO Accession GSM4686514 SRX8808588 GSM4686515 SRP273093 PRJNA647773 SRS7074634 GSM4686515 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686515 GSM4686515 GEO Accession GSM4686515 SRX8808589 GSM4686516 SRP273093 PRJNA647773 SRS7074637 GSM4686516 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686516 GSM4686516 GEO Accession GSM4686516 SRX8808590 GSM4686517 SRP273093 PRJNA647773 SRS7074636 GSM4686517 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686517 GSM4686517 GEO Accession GSM4686517 SRX8808591 GSM4686518 SRP273093 PRJNA647773 SRS7074639 GSM4686518 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686518 GSM4686518 GEO Accession GSM4686518 SRX8808592 GSM4686519 SRP273093 PRJNA647773 SRS7074638 GSM4686519 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686519 GSM4686519 GEO Accession GSM4686519 SRX8808593 GSM4686520 SRP273093 PRJNA647773 SRS7074640 GSM4686520 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686520 GSM4686520 GEO Accession GSM4686520 SRX8808594 GSM4686521 SRP273093 PRJNA647773 SRS7074641 GSM4686521 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686521 GSM4686521 GEO Accession GSM4686521 SRX8808595 GSM4686522 SRP273093 PRJNA647773 SRS7074642 GSM4686522 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686522 GSM4686522 GEO Accession GSM4686522 SRX8808596 GSM4686523 SRP273093 PRJNA647773 SRS7074643 GSM4686523 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686523 GSM4686523 GEO Accession GSM4686523 SRX8808597 GSM4686524 SRP273093 PRJNA647773 SRS7074644 GSM4686524 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686524 GSM4686524 GEO Accession GSM4686524 SRX8808598 GSM4686525 SRP273093 PRJNA647773 SRS7074645 GSM4686525 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686525 GSM4686525 GEO Accession GSM4686525 SRX8808599 GSM4686526 SRP273093 PRJNA647773 SRS7074646 GSM4686526 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686526 GSM4686526 GEO Accession GSM4686526 SRX8808600 GSM4686527 SRP273093 PRJNA647773 SRS7074647 GSM4686527 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686527 GSM4686527 GEO Accession GSM4686527 SRX8808601 GSM4686528 SRP273093 PRJNA647773 SRS7074648 GSM4686528 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686528 GSM4686528 GEO Accession GSM4686528 SRX8808602 GSM4686529 SRP273093 PRJNA647773 SRS7074649 GSM4686529 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686529 GSM4686529 GEO Accession GSM4686529 SRX8808603 GSM4686530 SRP273093 PRJNA647773 SRS7074650 GSM4686530 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686530 GSM4686530 GEO Accession GSM4686530 SRX8808604 GSM4686531 SRP273093 PRJNA647773 SRS7074651 GSM4686531 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686531 GSM4686531 GEO Accession GSM4686531 SRX8808605 GSM4686532 SRP273093 PRJNA647773 SRS7074652 GSM4686532 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686532 GSM4686532 GEO Accession GSM4686532 SRX8808606 GSM4686533 SRP273093 PRJNA647773 SRS7074653 GSM4686533 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686533 GSM4686533 GEO Accession GSM4686533 SRX8808607 GSM4686534 SRP273093 PRJNA647773 SRS7074654 GSM4686534 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686534 GSM4686534 GEO Accession GSM4686534 SRX8808608 GSM4686535 SRP273093 PRJNA647773 SRS7074656 GSM4686535 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686535 GSM4686535 GEO Accession GSM4686535 SRX8808609 GSM4686536 SRP273093 PRJNA647773 SRS7074655 GSM4686536 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686536 GSM4686536 GEO Accession GSM4686536 SRX8808610 GSM4686537 SRP273093 PRJNA647773 SRS7074657 GSM4686537 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686537 GSM4686537 GEO Accession GSM4686537 SRX8808611 GSM4686538 SRP273093 PRJNA647773 SRS7074658 GSM4686538 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686538 GSM4686538 GEO Accession GSM4686538 SRX8808612 GSM4686539 SRP273093 PRJNA647773 SRS7074659 GSM4686539 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686539 GSM4686539 GEO Accession GSM4686539 SRX8808613 GSM4686540 SRP273093 PRJNA647773 SRS7074660 GSM4686540 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686540 GSM4686540 GEO Accession GSM4686540 SRX8808614 GSM4686541 SRP273093 PRJNA647773 SRS7074661 GSM4686541 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686541 GSM4686541 GEO Accession GSM4686541 SRX8808615 GSM4686542 SRP273093 PRJNA647773 SRS7074664 GSM4686542 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686542 GSM4686542 GEO Accession GSM4686542 SRX8808616 GSM4686543 SRP273093 PRJNA647773 SRS7074662 GSM4686543 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686543 GSM4686543 GEO Accession GSM4686543 SRX8808617 GSM4686544 SRP273093 PRJNA647773 SRS7074663 GSM4686544 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686544 GSM4686544 GEO Accession GSM4686544 SRX8808618 GSM4686545 SRP273093 PRJNA647773 SRS7074665 GSM4686545 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686545 GSM4686545 GEO Accession GSM4686545 SRX8808619 GSM4686546 SRP273093 PRJNA647773 SRS7074666 GSM4686546 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686546 GSM4686546 GEO Accession GSM4686546 SRX8808620 GSM4686547 SRP273093 PRJNA647773 SRS7074667 GSM4686547 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686547 GSM4686547 GEO Accession GSM4686547 SRX8808621 GSM4686548 SRP273093 PRJNA647773 SRS7074668 GSM4686548 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686548 GSM4686548 GEO Accession GSM4686548 SRX8808622 GSM4686549 SRP273093 PRJNA647773 SRS7074671 GSM4686549 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686549 GSM4686549 GEO Accession GSM4686549 SRX8808623 GSM4686550 SRP273093 PRJNA647773 SRS7074669 GSM4686550 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686550 GSM4686550 GEO Accession GSM4686550 SRX8808624 GSM4686551 SRP273093 PRJNA647773 SRS7074670 GSM4686551 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686551 GSM4686551 GEO Accession GSM4686551 SRX8808625 GSM4686552 SRP273093 PRJNA647773 SRS7074672 GSM4686552 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686552 GSM4686552 GEO Accession GSM4686552 SRX8808626 GSM4686553 SRP273093 PRJNA647773 SRS7074673 GSM4686553 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686553 GSM4686553 GEO Accession GSM4686553 SRX8808627 GSM4686554 SRP273093 PRJNA647773 SRS7074674 GSM4686554 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686554 GSM4686554 GEO Accession GSM4686554 SRX8808628 GSM4686555 SRP273093 PRJNA647773 SRS7074675 GSM4686555 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686555 GSM4686555 GEO Accession GSM4686555 SRX8808629 GSM4686556 SRP273093 PRJNA647773 SRS7074677 GSM4686556 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686556 GSM4686556 GEO Accession GSM4686556 SRX8808630 GSM4686557 SRP273093 PRJNA647773 SRS7074678 GSM4686557 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686557 GSM4686557 GEO Accession GSM4686557 SRX8808631 GSM4686558 SRP273093 PRJNA647773 SRS7074676 GSM4686558 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686558 GSM4686558 GEO Accession GSM4686558 SRX8808632 GSM4686559 SRP273093 PRJNA647773 SRS7074679 GSM4686559 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686559 GSM4686559 GEO Accession GSM4686559 SRX8808633 GSM4686560 SRP273093 PRJNA647773 SRS7074680 GSM4686560 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686560 GSM4686560 GEO Accession GSM4686560 SRX8808634 GSM4686561 SRP273093 PRJNA647773 SRS7074681 GSM4686561 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686561 GSM4686561 GEO Accession GSM4686561 SRX8808635 GSM4686562 SRP273093 PRJNA647773 SRS7074684 GSM4686562 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686562 GSM4686562 GEO Accession GSM4686562 SRX8808636 GSM4686563 SRP273093 PRJNA647773 SRS7074682 GSM4686563 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686563 GSM4686563 GEO Accession GSM4686563 SRX8808637 GSM4686564 SRP273093 PRJNA647773 SRS7074683 GSM4686564 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686564 GSM4686564 GEO Accession GSM4686564 SRX8808638 GSM4686565 SRP273093 PRJNA647773 SRS7074685 GSM4686565 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686565 GSM4686565 GEO Accession GSM4686565 SRX8808639 GSM4686566 SRP273093 PRJNA647773 SRS7074687 GSM4686566 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686566 GSM4686566 GEO Accession GSM4686566 SRX8808640 GSM4686567 SRP273093 PRJNA647773 SRS7074686 GSM4686567 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686567 GSM4686567 GEO Accession GSM4686567 SRX8808641 GSM4686568 SRP273093 PRJNA647773 SRS7074688 GSM4686568 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686568 GSM4686568 GEO Accession GSM4686568 SRX8808642 GSM4686569 SRP273093 PRJNA647773 SRS7074690 GSM4686569 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686569 GSM4686569 GEO Accession GSM4686569 SRX8808643 GSM4686570 SRP273093 PRJNA647773 SRS7074689 GSM4686570 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686570 GSM4686570 GEO Accession GSM4686570 SRX8808644 GSM4686571 SRP273093 PRJNA647773 SRS7074692 GSM4686571 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686571 GSM4686571 GEO Accession GSM4686571 SRX8808645 GSM4686572 SRP273093 PRJNA647773 SRS7074691 GSM4686572 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686572 GSM4686572 GEO Accession GSM4686572 SRX8808646 GSM4686573 SRP273093 PRJNA647773 SRS7074693 GSM4686573 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686573 GSM4686573 GEO Accession GSM4686573 SRX8808647 GSM4686574 SRP273093 PRJNA647773 SRS7074694 GSM4686574 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686574 GSM4686574 GEO Accession GSM4686574 SRX8808648 GSM4686575 SRP273093 PRJNA647773 SRS7074697 GSM4686575 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686575 GSM4686575 GEO Accession GSM4686575 SRX8808649 GSM4686576 SRP273093 PRJNA647773 SRS7074695 GSM4686576 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686576 GSM4686576 GEO Accession GSM4686576 SRX8808650 GSM4686577 SRP273093 PRJNA647773 SRS7074696 GSM4686577 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686577 GSM4686577 GEO Accession GSM4686577 SRX8808651 GSM4686578 SRP273093 PRJNA647773 SRS7074698 GSM4686578 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686578 GSM4686578 GEO Accession GSM4686578 SRX8808652 GSM4686579 SRP273093 PRJNA647773 SRS7074699 GSM4686579 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686579 GSM4686579 GEO Accession GSM4686579 SRX8808653 GSM4686580 SRP273093 PRJNA647773 SRS7074700 GSM4686580 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686580 GSM4686580 GEO Accession GSM4686580 SRX8808654 GSM4686581 SRP273093 PRJNA647773 SRS7074701 GSM4686581 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686581 GSM4686581 GEO Accession GSM4686581 SRX8808655 GSM4686582 SRP273093 PRJNA647773 SRS7074703 GSM4686582 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686582 GSM4686582 GEO Accession GSM4686582 SRX8808656 GSM4686583 SRP273093 PRJNA647773 SRS7074702 GSM4686583 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686583 GSM4686583 GEO Accession GSM4686583 SRX8808657 GSM4686584 SRP273093 PRJNA647773 SRS7074704 GSM4686584 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686584 GSM4686584 GEO Accession GSM4686584 SRX8808658 GSM4686585 SRP273093 PRJNA647773 SRS7074705 GSM4686585 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686585 GSM4686585 GEO Accession GSM4686585 SRX8808659 GSM4686586 SRP273093 PRJNA647773 SRS7074706 GSM4686586 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686586 GSM4686586 GEO Accession GSM4686586 SRX8808660 GSM4686587 SRP273093 PRJNA647773 SRS7074707 GSM4686587 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686587 GSM4686587 GEO Accession GSM4686587 SRX8808661 GSM4686588 SRP273093 PRJNA647773 SRS7074708 GSM4686588 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686588 GSM4686588 GEO Accession GSM4686588 SRX8808662 GSM4686589 SRP273093 PRJNA647773 SRS7074709 GSM4686589 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686589 GSM4686589 GEO Accession GSM4686589 SRX8808663 GSM4686590 SRP273093 PRJNA647773 SRS7074710 GSM4686590 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686590 GSM4686590 GEO Accession GSM4686590 SRX8808664 GSM4686591 SRP273093 PRJNA647773 SRS7074711 GSM4686591 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686591 GSM4686591 GEO Accession GSM4686591 SRX8808665 GSM4686592 SRP273093 PRJNA647773 SRS7074712 GSM4686592 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686592 GSM4686592 GEO Accession GSM4686592 SRX8808666 GSM4686593 SRP273093 PRJNA647773 SRS7074714 GSM4686593 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686593 GSM4686593 GEO Accession GSM4686593 SRX8808667 GSM4686594 SRP273093 PRJNA647773 SRS7074713 GSM4686594 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686594 GSM4686594 GEO Accession GSM4686594 SRX8808668 GSM4686595 SRP273093 PRJNA647773 SRS7074715 GSM4686595 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686595 GSM4686595 GEO Accession GSM4686595 SRX8808669 GSM4686596 SRP273093 PRJNA647773 SRS7074716 GSM4686596 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686596 GSM4686596 GEO Accession GSM4686596 SRX8808670 GSM4686597 SRP273093 PRJNA647773 SRS7074717 GSM4686597 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686597 GSM4686597 GEO Accession GSM4686597 SRX8808671 GSM4686598 SRP273093 PRJNA647773 SRS7074718 GSM4686598 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686598 GSM4686598 GEO Accession GSM4686598 SRX8808672 GSM4686599 SRP273093 PRJNA647773 SRS7074719 GSM4686599 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686599 GSM4686599 GEO Accession GSM4686599 SRX8808673 GSM4686600 SRP273093 PRJNA647773 SRS7074720 GSM4686600 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686600 GSM4686600 GEO Accession GSM4686600 SRX8808674 GSM4686601 SRP273093 PRJNA647773 SRS7074721 GSM4686601 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686601 GSM4686601 GEO Accession GSM4686601 SRX8808675 GSM4686602 SRP273093 PRJNA647773 SRS7074724 GSM4686602 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686602 GSM4686602 GEO Accession GSM4686602 SRX8808676 GSM4686603 SRP273093 PRJNA647773 SRS7074722 GSM4686603 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686603 GSM4686603 GEO Accession GSM4686603 SRX8808677 GSM4686604 SRP273093 PRJNA647773 SRS7074723 GSM4686604 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686604 GSM4686604 GEO Accession GSM4686604 SRX8808678 GSM4686605 SRP273093 PRJNA647773 SRS7074725 GSM4686605 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686605 GSM4686605 GEO Accession GSM4686605 SRX8808679 GSM4686606 SRP273093 PRJNA647773 SRS7074726 GSM4686606 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686606 GSM4686606 GEO Accession GSM4686606 SRX8808680 GSM4686607 SRP273093 PRJNA647773 SRS7074727 GSM4686607 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686607 GSM4686607 GEO Accession GSM4686607 SRX8808681 GSM4686608 SRP273093 PRJNA647773 SRS7074728 GSM4686608 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686608 GSM4686608 GEO Accession GSM4686608 SRX8808682 GSM4686609 SRP273093 PRJNA647773 SRS7074729 GSM4686609 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686609 GSM4686609 GEO Accession GSM4686609 SRX8808683 GSM4686610 SRP273093 PRJNA647773 SRS7074732 GSM4686610 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686610 GSM4686610 GEO Accession GSM4686610 SRX8808684 GSM4686611 SRP273093 PRJNA647773 SRS7074730 GSM4686611 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686611 GSM4686611 GEO Accession GSM4686611 SRX8808685 GSM4686612 SRP273093 PRJNA647773 SRS7074731 GSM4686612 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686612 GSM4686612 GEO Accession GSM4686612 SRX8808686 GSM4686613 SRP273093 PRJNA647773 SRS7074733 GSM4686613 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686613 GSM4686613 GEO Accession GSM4686613 SRX8808687 GSM4686614 SRP273093 PRJNA647773 SRS7074734 GSM4686614 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686614 GSM4686614 GEO Accession GSM4686614 SRX8808688 GSM4686615 SRP273093 PRJNA647773 SRS7074735 GSM4686615 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686615 GSM4686615 GEO Accession GSM4686615 SRX8808689 GSM4686616 SRP273093 PRJNA647773 SRS7074736 GSM4686616 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686616 GSM4686616 GEO Accession GSM4686616 SRX8808690 GSM4686617 SRP273093 PRJNA647773 SRS7074738 GSM4686617 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686617 GSM4686617 GEO Accession GSM4686617 SRX8808691 GSM4686618 SRP273093 PRJNA647773 SRS7074737 GSM4686618 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686618 GSM4686618 GEO Accession GSM4686618 SRX8808692 GSM4686619 SRP273093 PRJNA647773 SRS7074739 GSM4686619 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686619 GSM4686619 GEO Accession GSM4686619 SRX8808693 GSM4686620 SRP273093 PRJNA647773 SRS7074740 GSM4686620 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686620 GSM4686620 GEO Accession GSM4686620 SRX8808694 GSM4686621 SRP273093 PRJNA647773 SRS7074741 GSM4686621 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686621 GSM4686621 GEO Accession GSM4686621 SRX8808695 GSM4686622 SRP273093 PRJNA647773 SRS7074742 GSM4686622 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686622 GSM4686622 GEO Accession GSM4686622 SRX8808696 GSM4686623 SRP273093 PRJNA647773 SRS7074743 GSM4686623 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686623 GSM4686623 GEO Accession GSM4686623 SRX8808697 GSM4686624 SRP273093 PRJNA647773 SRS7074746 GSM4686624 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686624 GSM4686624 GEO Accession GSM4686624 SRX8808698 GSM4686625 SRP273093 PRJNA647773 SRS7074744 GSM4686625 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686625 GSM4686625 GEO Accession GSM4686625 SRX8808699 GSM4686626 SRP273093 PRJNA647773 SRS7074745 GSM4686626 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686626 GSM4686626 GEO Accession GSM4686626 SRX8808700 GSM4686627 SRP273093 PRJNA647773 SRS7074747 GSM4686627 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686627 GSM4686627 GEO Accession GSM4686627 SRX8808701 GSM4686628 SRP273093 PRJNA647773 SRS7074748 GSM4686628 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686628 GSM4686628 GEO Accession GSM4686628 SRX8808702 GSM4686629 SRP273093 PRJNA647773 SRS7074749 GSM4686629 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686629 GSM4686629 GEO Accession GSM4686629 SRX8808703 GSM4686630 SRP273093 PRJNA647773 SRS7074750 GSM4686630 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686630 GSM4686630 GEO Accession GSM4686630 SRX8808704 GSM4686631 SRP273093 PRJNA647773 SRS7074751 GSM4686631 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686631 GSM4686631 GEO Accession GSM4686631 SRX8808705 GSM4686632 SRP273093 PRJNA647773 SRS7074752 GSM4686632 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686632 GSM4686632 GEO Accession GSM4686632 SRX8808706 GSM4686633 SRP273093 PRJNA647773 SRS7074754 GSM4686633 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686633 GSM4686633 GEO Accession GSM4686633 SRX8808707 GSM4686634 SRP273093 PRJNA647773 SRS7074753 GSM4686634 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686634 GSM4686634 GEO Accession GSM4686634 SRX8808708 GSM4686635 SRP273093 PRJNA647773 SRS7074755 GSM4686635 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686635 GSM4686635 GEO Accession GSM4686635 SRX8808709 GSM4686636 SRP273093 PRJNA647773 SRS7074756 GSM4686636 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686636 GSM4686636 GEO Accession GSM4686636 SRX8808710 GSM4686637 SRP273093 PRJNA647773 SRS7074758 GSM4686637 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686637 GSM4686637 GEO Accession GSM4686637 SRX8808711 GSM4686638 SRP273093 PRJNA647773 SRS7074757 GSM4686638 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686638 GSM4686638 GEO Accession GSM4686638 SRX8808712 GSM4686639 SRP273093 PRJNA647773 SRS7074759 GSM4686639 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686639 GSM4686639 GEO Accession GSM4686639 SRX8808713 GSM4686640 SRP273093 PRJNA647773 SRS7074760 GSM4686640 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686640 GSM4686640 GEO Accession GSM4686640 SRX8808714 GSM4686641 SRP273093 PRJNA647773 SRS7074761 GSM4686641 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686641 GSM4686641 GEO Accession GSM4686641 SRX8808715 GSM4686642 SRP273093 PRJNA647773 SRS7074762 GSM4686642 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686642 GSM4686642 GEO Accession GSM4686642 SRX8808716 GSM4686643 SRP273093 PRJNA647773 SRS7074763 GSM4686643 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686643 GSM4686643 GEO Accession GSM4686643 SRX8808717 GSM4686644 SRP273093 PRJNA647773 SRS7074764 GSM4686644 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686644 GSM4686644 GEO Accession GSM4686644 SRX8808718 GSM4686645 SRP273093 PRJNA647773 SRS7074766 GSM4686645 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686645 GSM4686645 GEO Accession GSM4686645 SRX8808719 GSM4686646 SRP273093 PRJNA647773 SRS7074765 GSM4686646 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686646 GSM4686646 GEO Accession GSM4686646 SRX8808720 GSM4686647 SRP273093 PRJNA647773 SRS7074767 GSM4686647 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686647 GSM4686647 GEO Accession GSM4686647 SRX8808721 GSM4686648 SRP273093 PRJNA647773 SRS7074768 GSM4686648 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686648 GSM4686648 GEO Accession GSM4686648 SRX8808722 GSM4686649 SRP273093 PRJNA647773 SRS7074769 GSM4686649 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686649 GSM4686649 GEO Accession GSM4686649 SRX8808723 GSM4686650 SRP273093 PRJNA647773 SRS7074770 GSM4686650 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686650 GSM4686650 GEO Accession GSM4686650 SRX8808724 GSM4686651 SRP273093 PRJNA647773 SRS7074772 GSM4686651 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686651 GSM4686651 GEO Accession GSM4686651 SRX8808725 GSM4686652 SRP273093 PRJNA647773 SRS7074771 GSM4686652 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686652 GSM4686652 GEO Accession GSM4686652 SRX8808726 GSM4686653 SRP273093 PRJNA647773 SRS7074773 GSM4686653 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686653 GSM4686653 GEO Accession GSM4686653 SRX8808727 GSM4686654 SRP273093 PRJNA647773 SRS7074774 GSM4686654 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686654 GSM4686654 GEO Accession GSM4686654 SRX8808728 GSM4686655 SRP273093 PRJNA647773 SRS7074775 GSM4686655 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686655 GSM4686655 GEO Accession GSM4686655 SRX8808729 GSM4686656 SRP273093 PRJNA647773 SRS7074776 GSM4686656 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686656 GSM4686656 GEO Accession GSM4686656 SRX8808730 GSM4686657 SRP273093 PRJNA647773 SRS7074777 GSM4686657 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686657 GSM4686657 GEO Accession GSM4686657 SRX8808731 GSM4686658 SRP273093 PRJNA647773 SRS7074778 GSM4686658 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686658 GSM4686658 GEO Accession GSM4686658 SRX8808732 GSM4686659 SRP273093 PRJNA647773 SRS7074779 GSM4686659 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686659 GSM4686659 GEO Accession GSM4686659 SRX8808733 GSM4686660 SRP273093 PRJNA647773 SRS7074780 GSM4686660 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686660 GSM4686660 GEO Accession GSM4686660 SRX8808734 GSM4686661 SRP273093 PRJNA647773 SRS7074781 GSM4686661 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686661 GSM4686661 GEO Accession GSM4686661 SRX8808735 GSM4686662 SRP273093 PRJNA647773 SRS7074782 GSM4686662 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686662 GSM4686662 GEO Accession GSM4686662 SRX8808736 GSM4686663 SRP273093 PRJNA647773 SRS7074783 GSM4686663 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686663 GSM4686663 GEO Accession GSM4686663 SRX8808737 GSM4686664 SRP273093 PRJNA647773 SRS7074784 GSM4686664 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686664 GSM4686664 GEO Accession GSM4686664 SRX8808738 GSM4686665 SRP273093 PRJNA647773 SRS7074786 GSM4686665 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686665 GSM4686665 GEO Accession GSM4686665 SRX8808739 GSM4686666 SRP273093 PRJNA647773 SRS7074785 GSM4686666 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686666 GSM4686666 GEO Accession GSM4686666 SRX8808740 GSM4686667 SRP273093 PRJNA647773 SRS7074787 GSM4686667 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686667 GSM4686667 GEO Accession GSM4686667 SRX8808741 GSM4686668 SRP273093 PRJNA647773 SRS7074788 GSM4686668 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686668 GSM4686668 GEO Accession GSM4686668 SRX8808742 GSM4686669 SRP273093 PRJNA647773 SRS7074789 GSM4686669 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686669 GSM4686669 GEO Accession GSM4686669 SRX8808743 GSM4686670 SRP273093 PRJNA647773 SRS7074790 GSM4686670 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686670 GSM4686670 GEO Accession GSM4686670 SRX8808744 GSM4686671 SRP273093 PRJNA647773 SRS7074791 GSM4686671 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686671 GSM4686671 GEO Accession GSM4686671 SRX8808745 GSM4686672 SRP273093 PRJNA647773 SRS7074793 GSM4686672 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686672 GSM4686672 GEO Accession GSM4686672 SRX8808746 GSM4686673 SRP273093 PRJNA647773 SRS7074794 GSM4686673 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686673 GSM4686673 GEO Accession GSM4686673 SRX8808747 GSM4686674 SRP273093 PRJNA647773 SRS7074792 GSM4686674 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686674 GSM4686674 GEO Accession GSM4686674 SRX8808748 GSM4686675 SRP273093 PRJNA647773 SRS7074795 GSM4686675 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686675 GSM4686675 GEO Accession GSM4686675 SRX8808749 GSM4686676 SRP273093 PRJNA647773 SRS7074796 GSM4686676 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686676 GSM4686676 GEO Accession GSM4686676 SRX8808750 GSM4686677 SRP273093 PRJNA647773 SRS7074797 GSM4686677 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686677 GSM4686677 GEO Accession GSM4686677 SRX8808751 GSM4686678 SRP273093 PRJNA647773 SRS7074798 GSM4686678 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686678 GSM4686678 GEO Accession GSM4686678 SRX8808752 GSM4686679 SRP273093 PRJNA647773 SRS7074800 GSM4686679 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686679 GSM4686679 GEO Accession GSM4686679 SRX8808753 GSM4686680 SRP273093 PRJNA647773 SRS7074799 GSM4686680 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686680 GSM4686680 GEO Accession GSM4686680 SRX8808754 GSM4686681 SRP273093 PRJNA647773 SRS7074801 GSM4686681 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686681 GSM4686681 GEO Accession GSM4686681 SRX8808755 GSM4686682 SRP273093 PRJNA647773 SRS7074802 GSM4686682 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686682 GSM4686682 GEO Accession GSM4686682 SRX8808756 GSM4686683 SRP273093 PRJNA647773 SRS7074803 GSM4686683 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686683 GSM4686683 GEO Accession GSM4686683 SRX8808757 GSM4686684 SRP273093 PRJNA647773 SRS7074804 GSM4686684 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686684 GSM4686684 GEO Accession GSM4686684 SRX8808758 GSM4686685 SRP273093 PRJNA647773 SRS7074805 GSM4686685 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686685 GSM4686685 GEO Accession GSM4686685 SRX8808759 GSM4686686 SRP273093 PRJNA647773 SRS7074807 GSM4686686 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686686 GSM4686686 GEO Accession GSM4686686 SRX8808760 GSM4686687 SRP273093 PRJNA647773 SRS7074806 GSM4686687 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686687 GSM4686687 GEO Accession GSM4686687 SRX8808761 GSM4686688 SRP273093 PRJNA647773 SRS7074808 GSM4686688 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686688 GSM4686688 GEO Accession GSM4686688 SRX8808762 GSM4686689 SRP273093 PRJNA647773 SRS7074809 GSM4686689 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686689 GSM4686689 GEO Accession GSM4686689 SRX8808763 GSM4686690 SRP273093 PRJNA647773 SRS7074810 GSM4686690 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686690 GSM4686690 GEO Accession GSM4686690 SRX8808764 GSM4686691 SRP273093 PRJNA647773 SRS7074811 GSM4686691 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686691 GSM4686691 GEO Accession GSM4686691 SRX8808765 GSM4686692 SRP273093 PRJNA647773 SRS7074812 GSM4686692 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686692 GSM4686692 GEO Accession GSM4686692 SRX8808766 GSM4686693 SRP273093 PRJNA647773 SRS7074814 GSM4686693 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686693 GSM4686693 GEO Accession GSM4686693 SRX8808767 GSM4686694 SRP273093 PRJNA647773 SRS7074813 GSM4686694 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686694 GSM4686694 GEO Accession GSM4686694 SRX8808768 GSM4686695 SRP273093 PRJNA647773 SRS7074815 GSM4686695 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686695 GSM4686695 GEO Accession GSM4686695 SRX8808769 GSM4686696 SRP273093 PRJNA647773 SRS7074816 GSM4686696 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686696 GSM4686696 GEO Accession GSM4686696 SRX8808770 GSM4686697 SRP273093 PRJNA647773 SRS7074817 GSM4686697 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686697 GSM4686697 GEO Accession GSM4686697 SRX8808771 GSM4686698 SRP273093 PRJNA647773 SRS7074818 GSM4686698 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686698 GSM4686698 GEO Accession GSM4686698 SRX8808772 GSM4686699 SRP273093 PRJNA647773 SRS7074820 GSM4686699 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686699 GSM4686699 GEO Accession GSM4686699 SRX8808773 GSM4686700 SRP273093 PRJNA647773 SRS7074819 GSM4686700 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686700 GSM4686700 GEO Accession GSM4686700 SRX8808774 GSM4686701 SRP273093 PRJNA647773 SRS7074821 GSM4686701 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686701 GSM4686701 GEO Accession GSM4686701 SRX8808775 GSM4686702 SRP273093 PRJNA647773 SRS7074822 GSM4686702 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686702 GSM4686702 GEO Accession GSM4686702 SRX8808776 GSM4686703 SRP273093 PRJNA647773 SRS7074823 GSM4686703 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686703 GSM4686703 GEO Accession GSM4686703 SRX8808777 GSM4686704 SRP273093 PRJNA647773 SRS7074824 GSM4686704 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686704 GSM4686704 GEO Accession GSM4686704 SRX8808778 GSM4686705 SRP273093 PRJNA647773 SRS7074825 GSM4686705 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686705 GSM4686705 GEO Accession GSM4686705 SRX8808779 GSM4686706 SRP273093 PRJNA647773 SRS7074826 GSM4686706 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686706 GSM4686706 GEO Accession GSM4686706 SRX8808780 GSM4686707 SRP273093 PRJNA647773 SRS7074827 GSM4686707 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686707 GSM4686707 GEO Accession GSM4686707 SRX8808781 GSM4686708 SRP273093 PRJNA647773 SRS7074828 GSM4686708 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686708 GSM4686708 GEO Accession GSM4686708 SRX8808782 GSM4686709 SRP273093 PRJNA647773 SRS7074829 GSM4686709 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686709 GSM4686709 GEO Accession GSM4686709 SRX8808783 GSM4686710 SRP273093 PRJNA647773 SRS7074830 GSM4686710 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686710 GSM4686710 GEO Accession GSM4686710 SRX8808784 GSM4686711 SRP273093 PRJNA647773 SRS7074831 GSM4686711 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686711 GSM4686711 GEO Accession GSM4686711 SRX8808785 GSM4686712 SRP273093 PRJNA647773 SRS7074832 GSM4686712 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686712 GSM4686712 GEO Accession GSM4686712 SRX8808786 GSM4686713 SRP273093 PRJNA647773 SRS7074834 GSM4686713 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686713 GSM4686713 GEO Accession GSM4686713 SRX8808787 GSM4686714 SRP273093 PRJNA647773 SRS7074833 GSM4686714 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686714 GSM4686714 GEO Accession GSM4686714 SRX8808788 GSM4686715 SRP273093 PRJNA647773 SRS7074835 GSM4686715 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686715 GSM4686715 GEO Accession GSM4686715 SRX8808789 GSM4686716 SRP273093 PRJNA647773 SRS7074836 GSM4686716 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686716 GSM4686716 GEO Accession GSM4686716 SRX8808790 GSM4686717 SRP273093 PRJNA647773 SRS7074837 GSM4686717 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686717 GSM4686717 GEO Accession GSM4686717 SRX8808791 GSM4686718 SRP273093 PRJNA647773 SRS7074838 GSM4686718 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686718 GSM4686718 GEO Accession GSM4686718 SRX8808792 GSM4686719 SRP273093 PRJNA647773 SRS7074839 GSM4686719 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686719 GSM4686719 GEO Accession GSM4686719 SRX8808793 GSM4686720 SRP273093 PRJNA647773 SRS7074841 GSM4686720 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686720 GSM4686720 GEO Accession GSM4686720 SRX8808794 GSM4686721 SRP273093 PRJNA647773 SRS7074840 GSM4686721 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686721 GSM4686721 GEO Accession GSM4686721 SRX8808795 GSM4686722 SRP273093 PRJNA647773 SRS7074842 GSM4686722 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686722 GSM4686722 GEO Accession GSM4686722 SRX8808796 GSM4686723 SRP273093 PRJNA647773 SRS7074843 GSM4686723 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686723 GSM4686723 GEO Accession GSM4686723 SRX8808797 GSM4686724 SRP273093 PRJNA647773 SRS7074844 GSM4686724 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686724 GSM4686724 GEO Accession GSM4686724 SRX8808798 GSM4686725 SRP273093 PRJNA647773 SRS7074845 GSM4686725 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686725 GSM4686725 GEO Accession GSM4686725 SRX8808799 GSM4686726 SRP273093 PRJNA647773 SRS7074848 GSM4686726 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686726 GSM4686726 GEO Accession GSM4686726 SRX8808800 GSM4686727 SRP273093 PRJNA647773 SRS7074846 GSM4686727 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686727 GSM4686727 GEO Accession GSM4686727 SRX8808801 GSM4686728 SRP273093 PRJNA647773 SRS7074847 GSM4686728 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686728 GSM4686728 GEO Accession GSM4686728 SRX8808802 GSM4686729 SRP273093 PRJNA647773 SRS7074849 GSM4686729 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686729 GSM4686729 GEO Accession GSM4686729 SRX8808803 GSM4686730 SRP273093 PRJNA647773 SRS7074850 GSM4686730 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686730 GSM4686730 GEO Accession GSM4686730 SRX8808804 GSM4686731 SRP273093 PRJNA647773 SRS7074851 GSM4686731 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686731 GSM4686731 GEO Accession GSM4686731 SRX8808805 GSM4686732 SRP273093 PRJNA647773 SRS7074853 GSM4686732 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686732 GSM4686732 GEO Accession GSM4686732 SRX8808806 GSM4686733 SRP273093 PRJNA647773 SRS7074852 GSM4686733 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686733 GSM4686733 GEO Accession GSM4686733 SRX8808807 GSM4686734 SRP273093 PRJNA647773 SRS7074854 GSM4686734 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686734 GSM4686734 GEO Accession GSM4686734 SRX8808808 GSM4686735 SRP273093 PRJNA647773 SRS7074855 GSM4686735 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686735 GSM4686735 GEO Accession GSM4686735 SRX8808809 GSM4686736 SRP273093 PRJNA647773 SRS7074856 GSM4686736 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686736 GSM4686736 GEO Accession GSM4686736 SRX8808810 GSM4686737 SRP273093 PRJNA647773 SRS7074857 GSM4686737 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686737 GSM4686737 GEO Accession GSM4686737 SRX8808811 GSM4686738 SRP273093 PRJNA647773 SRS7074859 GSM4686738 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686738 GSM4686738 GEO Accession GSM4686738 SRX8808812 GSM4686739 SRP273093 PRJNA647773 SRS7074858 GSM4686739 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686739 GSM4686739 GEO Accession GSM4686739 SRX8808813 GSM4686740 SRP273093 PRJNA647773 SRS7074860 GSM4686740 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686740 GSM4686740 GEO Accession GSM4686740 SRX8808814 GSM4686741 SRP273093 PRJNA647773 SRS7074861 GSM4686741 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686741 GSM4686741 GEO Accession GSM4686741 SRX8808815 GSM4686742 SRP273093 PRJNA647773 SRS7074862 GSM4686742 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686742 GSM4686742 GEO Accession GSM4686742 SRX8808816 GSM4686743 SRP273093 PRJNA647773 SRS7074863 GSM4686743 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686743 GSM4686743 GEO Accession GSM4686743 SRX8808817 GSM4686744 SRP273093 PRJNA647773 SRS7074865 GSM4686744 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686744 GSM4686744 GEO Accession GSM4686744 SRX8808818 GSM4686745 SRP273093 PRJNA647773 SRS7074864 GSM4686745 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686745 GSM4686745 GEO Accession GSM4686745 SRX8808819 GSM4686746 SRP273093 PRJNA647773 SRS7074866 GSM4686746 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686746 GSM4686746 GEO Accession GSM4686746 SRX8808820 GSM4686747 SRP273093 PRJNA647773 SRS7074867 GSM4686747 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686747 GSM4686747 GEO Accession GSM4686747 SRX8808821 GSM4686748 SRP273093 PRJNA647773 SRS7074868 GSM4686748 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686748 GSM4686748 GEO Accession GSM4686748 SRX8808822 GSM4686749 SRP273093 PRJNA647773 SRS7074870 GSM4686749 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686749 GSM4686749 GEO Accession GSM4686749 SRX8808823 GSM4686750 SRP273093 PRJNA647773 SRS7074869 GSM4686750 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686750 GSM4686750 GEO Accession GSM4686750 SRX8808824 GSM4686751 SRP273093 PRJNA647773 SRS7074871 GSM4686751 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686751 GSM4686751 GEO Accession GSM4686751 SRX8808825 GSM4686752 SRP273093 PRJNA647773 SRS7074872 GSM4686752 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686752 GSM4686752 GEO Accession GSM4686752 SRX8808826 GSM4686753 SRP273093 PRJNA647773 SRS7074873 GSM4686753 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686753 GSM4686753 GEO Accession GSM4686753 SRX8808827 GSM4686754 SRP273093 PRJNA647773 SRS7074874 GSM4686754 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686754 GSM4686754 GEO Accession GSM4686754 SRX8808828 GSM4686755 SRP273093 PRJNA647773 SRS7074875 GSM4686755 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686755 GSM4686755 GEO Accession GSM4686755 SRX8808829 GSM4686756 SRP273093 PRJNA647773 SRS7074876 GSM4686756 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686756 GSM4686756 GEO Accession GSM4686756 SRX8808830 GSM4686757 SRP273093 PRJNA647773 SRS7074877 GSM4686757 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686757 GSM4686757 GEO Accession GSM4686757 SRX8808831 GSM4686758 SRP273093 PRJNA647773 SRS7074878 GSM4686758 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686758 GSM4686758 GEO Accession GSM4686758 SRX8808832 GSM4686759 SRP273093 PRJNA647773 SRS7074879 GSM4686759 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686759 GSM4686759 GEO Accession GSM4686759 SRX8808833 GSM4686760 SRP273093 PRJNA647773 SRS7074880 GSM4686760 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686760 GSM4686760 GEO Accession GSM4686760 SRX8808834 GSM4686761 SRP273093 PRJNA647773 SRS7074881 GSM4686761 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686761 GSM4686761 GEO Accession GSM4686761 SRX8808835 GSM4686762 SRP273093 PRJNA647773 SRS7074882 GSM4686762 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686762 GSM4686762 GEO Accession GSM4686762 SRX8808836 GSM4686763 SRP273093 PRJNA647773 SRS7074884 GSM4686763 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686763 GSM4686763 GEO Accession GSM4686763 SRX8808837 GSM4686764 SRP273093 PRJNA647773 SRS7074883 GSM4686764 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686764 GSM4686764 GEO Accession GSM4686764 SRX8808838 GSM4686765 SRP273093 PRJNA647773 SRS7074886 GSM4686765 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686765 GSM4686765 GEO Accession GSM4686765 SRX8808839 GSM4686766 SRP273093 PRJNA647773 SRS7074885 GSM4686766 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686766 GSM4686766 GEO Accession GSM4686766 SRX8808840 GSM4686767 SRP273093 PRJNA647773 SRS7074887 GSM4686767 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686767 GSM4686767 GEO Accession GSM4686767 SRX8808841 GSM4686768 SRP273093 PRJNA647773 SRS7074888 GSM4686768 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686768 GSM4686768 GEO Accession GSM4686768 SRX8808842 GSM4686769 SRP273093 PRJNA647773 SRS7074889 GSM4686769 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686769 GSM4686769 GEO Accession GSM4686769 SRX8808843 GSM4686770 SRP273093 PRJNA647773 SRS7074890 GSM4686770 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686770 GSM4686770 GEO Accession GSM4686770 SRX8808844 GSM4686771 SRP273093 PRJNA647773 SRS7074892 GSM4686771 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686771 GSM4686771 GEO Accession GSM4686771 SRX8808845 GSM4686772 SRP273093 PRJNA647773 SRS7074891 GSM4686772 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686772 GSM4686772 GEO Accession GSM4686772 SRX8808846 GSM4686773 SRP273093 PRJNA647773 SRS7074894 GSM4686773 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686773 GSM4686773 GEO Accession GSM4686773 SRX8808847 GSM4686774 SRP273093 PRJNA647773 SRS7074893 GSM4686774 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686774 GSM4686774 GEO Accession GSM4686774 SRX8808849 GSM4686776 SRP273093 PRJNA647773 SRS7074896 GSM4686776 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686776 GSM4686776 GEO Accession GSM4686776 SRX8808850 GSM4686777 SRP273093 PRJNA647773 SRS7074897 GSM4686777 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686777 GSM4686777 GEO Accession GSM4686777 SRX8808851 GSM4686778 SRP273093 PRJNA647773 SRS7074899 GSM4686778 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686778 GSM4686778 GEO Accession GSM4686778 SRX8808852 GSM4686779 SRP273093 PRJNA647773 SRS7074898 GSM4686779 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686779 GSM4686779 GEO Accession GSM4686779 SRX8808853 GSM4686780 SRP273093 PRJNA647773 SRS7074900 GSM4686780 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686780 GSM4686780 GEO Accession GSM4686780 SRX8808854 GSM4686781 SRP273093 PRJNA647773 SRS7074901 GSM4686781 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686781 GSM4686781 GEO Accession GSM4686781 SRX8808855 GSM4686782 SRP273093 PRJNA647773 SRS7074903 GSM4686782 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686782 GSM4686782 GEO Accession GSM4686782 SRX8808856 GSM4686783 SRP273093 PRJNA647773 SRS7074904 GSM4686783 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686783 GSM4686783 GEO Accession GSM4686783 SRX8808857 GSM4686784 SRP273093 PRJNA647773 SRS7074907 GSM4686784 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686784 GSM4686784 GEO Accession GSM4686784 SRX8808858 GSM4686785 SRP273093 PRJNA647773 SRS7074902 GSM4686785 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686785 GSM4686785 GEO Accession GSM4686785 SRX8808859 GSM4686786 SRP273093 PRJNA647773 SRS7074908 GSM4686786 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686786 GSM4686786 GEO Accession GSM4686786 SRX8808860 GSM4686787 SRP273093 PRJNA647773 SRS7074905 GSM4686787 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686787 GSM4686787 GEO Accession GSM4686787 SRX8808861 GSM4686788 SRP273093 PRJNA647773 SRS7074906 GSM4686788 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686788 GSM4686788 GEO Accession GSM4686788 SRX8808862 GSM4686789 SRP273093 PRJNA647773 SRS7074910 GSM4686789 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686789 GSM4686789 GEO Accession GSM4686789 SRX8808863 GSM4686790 SRP273093 PRJNA647773 SRS7074909 GSM4686790 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686790 GSM4686790 GEO Accession GSM4686790 SRX8808864 GSM4686791 SRP273093 PRJNA647773 SRS7074911 GSM4686791 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686791 GSM4686791 GEO Accession GSM4686791 SRX8808865 GSM4686792 SRP273093 PRJNA647773 SRS7074913 GSM4686792 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686792 GSM4686792 GEO Accession GSM4686792 SRX8808866 GSM4686793 SRP273093 PRJNA647773 SRS7074912 GSM4686793 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686793 GSM4686793 GEO Accession GSM4686793 SRX8808867 GSM4686794 SRP273093 PRJNA647773 SRS7074914 GSM4686794 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686794 GSM4686794 GEO Accession GSM4686794 SRX8808868 GSM4686795 SRP273093 PRJNA647773 SRS7074915 GSM4686795 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686795 GSM4686795 GEO Accession GSM4686795 SRX8808869 GSM4688116 SRP273093 PRJNA647773 SRS7076092 GSM4688116 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688116 GSM4688116 GEO Accession GSM4688116 SRX8808870 GSM4688117 SRP273093 PRJNA647773 SRS7076093 GSM4688117 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688117 GSM4688117 GEO Accession GSM4688117 SRX8808871 GSM4688118 SRP273093 PRJNA647773 SRS7076094 GSM4688118 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688118 GSM4688118 GEO Accession GSM4688118 SRX8808872 GSM4688119 SRP273093 PRJNA647773 SRS7076095 GSM4688119 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688119 GSM4688119 GEO Accession GSM4688119 SRX8808873 GSM4688120 SRP273093 PRJNA647773 SRS7076097 GSM4688120 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688120 GSM4688120 GEO Accession GSM4688120 SRX8808874 GSM4688121 SRP273093 PRJNA647773 SRS7076096 GSM4688121 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688121 GSM4688121 GEO Accession GSM4688121 SRX8808875 GSM4688122 SRP273093 PRJNA647773 SRS7076098 GSM4688122 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688122 GSM4688122 GEO Accession GSM4688122 SRX8808876 GSM4688123 SRP273093 PRJNA647773 SRS7076099 GSM4688123 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688123 GSM4688123 GEO Accession GSM4688123 SRX8808877 GSM4688124 SRP273093 PRJNA647773 SRS7076100 GSM4688124 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688124 GSM4688124 GEO Accession GSM4688124 SRX8808878 GSM4688125 SRP273093 PRJNA647773 SRS7076101 GSM4688125 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688125 GSM4688125 GEO Accession GSM4688125 SRX8808879 GSM4688126 SRP273093 PRJNA647773 SRS7076102 GSM4688126 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688126 GSM4688126 GEO Accession GSM4688126 SRX8808880 GSM4688127 SRP273093 PRJNA647773 SRS7076104 GSM4688127 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688127 GSM4688127 GEO Accession GSM4688127 SRX8808881 GSM4688128 SRP273093 PRJNA647773 SRS7076103 GSM4688128 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688128 GSM4688128 GEO Accession GSM4688128 SRX8808882 GSM4688129 SRP273093 PRJNA647773 SRS7076106 GSM4688129 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688129 GSM4688129 GEO Accession GSM4688129 SRX8808883 GSM4688130 SRP273093 PRJNA647773 SRS7076105 GSM4688130 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688130 GSM4688130 GEO Accession GSM4688130 SRX8808884 GSM4688131 SRP273093 PRJNA647773 SRS7076108 GSM4688131 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688131 GSM4688131 GEO Accession GSM4688131 SRX8808885 GSM4688132 SRP273093 PRJNA647773 SRS7076107 GSM4688132 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688132 GSM4688132 GEO Accession GSM4688132 SRX8808886 GSM4688133 SRP273093 PRJNA647773 SRS7076111 GSM4688133 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688133 GSM4688133 GEO Accession GSM4688133 SRX8808887 GSM4688134 SRP273093 PRJNA647773 SRS7076109 GSM4688134 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688134 GSM4688134 GEO Accession GSM4688134 SRX8808888 GSM4688135 SRP273093 PRJNA647773 SRS7076110 GSM4688135 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688135 GSM4688135 GEO Accession GSM4688135 SRX8808889 GSM4688136 SRP273093 PRJNA647773 SRS7076112 GSM4688136 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688136 GSM4688136 GEO Accession GSM4688136 SRX8808890 GSM4688137 SRP273093 PRJNA647773 SRS7076114 GSM4688137 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688137 GSM4688137 GEO Accession GSM4688137 SRX8808891 GSM4688138 SRP273093 PRJNA647773 SRS7076113 GSM4688138 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688138 GSM4688138 GEO Accession GSM4688138 SRX8808892 GSM4688139 SRP273093 PRJNA647773 SRS7076115 GSM4688139 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688139 GSM4688139 GEO Accession GSM4688139 SRX8808893 GSM4688140 SRP273093 PRJNA647773 SRS7076116 GSM4688140 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688140 GSM4688140 GEO Accession GSM4688140 SRX8808894 GSM4688141 SRP273093 PRJNA647773 SRS7076117 GSM4688141 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688141 GSM4688141 GEO Accession GSM4688141 SRX8808895 GSM4688142 SRP273093 PRJNA647773 SRS7076118 GSM4688142 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688142 GSM4688142 GEO Accession GSM4688142 SRX8808896 GSM4688143 SRP273093 PRJNA647773 SRS7076120 GSM4688143 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688143 GSM4688143 GEO Accession GSM4688143 SRX8808897 GSM4688144 SRP273093 PRJNA647773 SRS7076119 GSM4688144 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688144 GSM4688144 GEO Accession GSM4688144 SRX8808898 GSM4688145 SRP273093 PRJNA647773 SRS7076121 GSM4688145 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688145 GSM4688145 GEO Accession GSM4688145 SRX8808899 GSM4688146 SRP273093 PRJNA647773 SRS7076122 GSM4688146 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688146 GSM4688146 GEO Accession GSM4688146 SRX8808900 GSM4688147 SRP273093 PRJNA647773 SRS7076124 GSM4688147 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688147 GSM4688147 GEO Accession GSM4688147 SRX8808901 GSM4688148 SRP273093 PRJNA647773 SRS7076123 GSM4688148 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688148 GSM4688148 GEO Accession GSM4688148 SRX8808902 GSM4688149 SRP273093 PRJNA647773 SRS7076127 GSM4688149 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688149 GSM4688149 GEO Accession GSM4688149 SRX8808903 GSM4688150 SRP273093 PRJNA647773 SRS7076125 GSM4688150 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688150 GSM4688150 GEO Accession GSM4688150 SRX8808904 GSM4688151 SRP273093 PRJNA647773 SRS7076126 GSM4688151 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688151 GSM4688151 GEO Accession GSM4688151 SRX8808905 GSM4688152 SRP273093 PRJNA647773 SRS7076128 GSM4688152 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688152 GSM4688152 GEO Accession GSM4688152 SRX8808906 GSM4688153 SRP273093 PRJNA647773 SRS7076129 GSM4688153 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688153 GSM4688153 GEO Accession GSM4688153 SRX8808907 GSM4688154 SRP273093 PRJNA647773 SRS7076130 GSM4688154 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688154 GSM4688154 GEO Accession GSM4688154 SRX8808908 GSM4688155 SRP273093 PRJNA647773 SRS7076132 GSM4688155 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688155 GSM4688155 GEO Accession GSM4688155 SRX8808909 GSM4688156 SRP273093 PRJNA647773 SRS7076131 GSM4688156 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688156 GSM4688156 GEO Accession GSM4688156 SRX8808910 GSM4688157 SRP273093 PRJNA647773 SRS7076133 GSM4688157 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688157 GSM4688157 GEO Accession GSM4688157 SRX8808911 GSM4688158 SRP273093 PRJNA647773 SRS7076134 GSM4688158 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688158 GSM4688158 GEO Accession GSM4688158 SRX8808912 GSM4688159 SRP273093 PRJNA647773 SRS7076135 GSM4688159 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688159 GSM4688159 GEO Accession GSM4688159 SRX8808913 GSM4688160 SRP273093 PRJNA647773 SRS7076136 GSM4688160 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688160 GSM4688160 GEO Accession GSM4688160 SRX8808914 GSM4688161 SRP273093 PRJNA647773 SRS7076137 GSM4688161 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688161 GSM4688161 GEO Accession GSM4688161 SRX8808915 GSM4688162 SRP273093 PRJNA647773 SRS7076138 GSM4688162 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688162 GSM4688162 GEO Accession GSM4688162 SRX8808916 GSM4688163 SRP273093 PRJNA647773 SRS7076139 GSM4688163 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688163 GSM4688163 GEO Accession GSM4688163 SRX8808917 GSM4688164 SRP273093 PRJNA647773 SRS7076140 GSM4688164 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688164 GSM4688164 GEO Accession GSM4688164 SRX8808918 GSM4688165 SRP273093 PRJNA647773 SRS7076141 GSM4688165 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688165 GSM4688165 GEO Accession GSM4688165 SRX8808919 GSM4688166 SRP273093 PRJNA647773 SRS7076142 GSM4688166 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688166 GSM4688166 GEO Accession GSM4688166 SRX8808920 GSM4688167 SRP273093 PRJNA647773 SRS7076143 GSM4688167 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688167 GSM4688167 GEO Accession GSM4688167 SRX8808921 GSM4688168 SRP273093 PRJNA647773 SRS7076144 GSM4688168 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688168 GSM4688168 GEO Accession GSM4688168 SRX8808922 GSM4688169 SRP273093 PRJNA647773 SRS7075297 GSM4688169 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688169 GSM4688169 GEO Accession GSM4688169 SRX8808923 GSM4688170 SRP273093 PRJNA647773 SRS7075299 GSM4688170 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688170 GSM4688170 GEO Accession GSM4688170 SRX8808924 GSM4688171 SRP273093 PRJNA647773 SRS7075298 GSM4688171 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688171 GSM4688171 GEO Accession GSM4688171 SRX8808925 GSM4688172 SRP273093 PRJNA647773 SRS7075300 GSM4688172 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688172 GSM4688172 GEO Accession GSM4688172 SRX8808926 GSM4688173 SRP273093 PRJNA647773 SRS7075301 GSM4688173 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688173 GSM4688173 GEO Accession GSM4688173 SRX8808927 GSM4688174 SRP273093 PRJNA647773 SRS7075302 GSM4688174 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688174 GSM4688174 GEO Accession GSM4688174 SRX8808928 GSM4688175 SRP273093 PRJNA647773 SRS7075303 GSM4688175 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688175 GSM4688175 GEO Accession GSM4688175 SRX8808929 GSM4688176 SRP273093 PRJNA647773 SRS7075306 GSM4688176 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688176 GSM4688176 GEO Accession GSM4688176 SRX8808930 GSM4688177 SRP273093 PRJNA647773 SRS7075304 GSM4688177 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688177 GSM4688177 GEO Accession GSM4688177 SRX8808931 GSM4688178 SRP273093 PRJNA647773 SRS7075305 GSM4688178 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688178 GSM4688178 GEO Accession GSM4688178 SRX8808932 GSM4688179 SRP273093 PRJNA647773 SRS7075307 GSM4688179 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688179 GSM4688179 GEO Accession GSM4688179 SRX8808933 GSM4688180 SRP273093 PRJNA647773 SRS7075308 GSM4688180 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688180 GSM4688180 GEO Accession GSM4688180 SRX8808934 GSM4688181 SRP273093 PRJNA647773 SRS7075309 GSM4688181 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688181 GSM4688181 GEO Accession GSM4688181 SRX8808935 GSM4688182 SRP273093 PRJNA647773 SRS7075310 GSM4688182 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688182 GSM4688182 GEO Accession GSM4688182 SRX8808936 GSM4688183 SRP273093 PRJNA647773 SRS7075311 GSM4688183 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688183 GSM4688183 GEO Accession GSM4688183 SRX8808937 GSM4688184 SRP273093 PRJNA647773 SRS7075312 GSM4688184 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688184 GSM4688184 GEO Accession GSM4688184 SRX8808938 GSM4688185 SRP273093 PRJNA647773 SRS7075313 GSM4688185 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688185 GSM4688185 GEO Accession GSM4688185 SRX8808939 GSM4688186 SRP273093 PRJNA647773 SRS7075316 GSM4688186 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688186 GSM4688186 GEO Accession GSM4688186 SRX8808940 GSM4688187 SRP273093 PRJNA647773 SRS7075314 GSM4688187 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688187 GSM4688187 GEO Accession GSM4688187 SRX8808941 GSM4688188 SRP273093 PRJNA647773 SRS7075315 GSM4688188 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688188 GSM4688188 GEO Accession GSM4688188 SRX8808942 GSM4688189 SRP273093 PRJNA647773 SRS7075317 GSM4688189 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688189 GSM4688189 GEO Accession GSM4688189 SRX8808943 GSM4688190 SRP273093 PRJNA647773 SRS7075318 GSM4688190 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688190 GSM4688190 GEO Accession GSM4688190 SRX8808944 GSM4688191 SRP273093 PRJNA647773 SRS7075319 GSM4688191 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688191 GSM4688191 GEO Accession GSM4688191 SRX8808945 GSM4688192 SRP273093 PRJNA647773 SRS7075320 GSM4688192 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688192 GSM4688192 GEO Accession GSM4688192 SRX8808946 GSM4688193 SRP273093 PRJNA647773 SRS7075321 GSM4688193 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688193 GSM4688193 GEO Accession GSM4688193 SRX8808947 GSM4688194 SRP273093 PRJNA647773 SRS7075322 GSM4688194 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688194 GSM4688194 GEO Accession GSM4688194 SRX8808948 GSM4688195 SRP273093 PRJNA647773 SRS7075323 GSM4688195 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688195 GSM4688195 GEO Accession GSM4688195 SRX8808949 GSM4688196 SRP273093 PRJNA647773 SRS7075324 GSM4688196 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688196 GSM4688196 GEO Accession GSM4688196 SRX8808950 GSM4688197 SRP273093 PRJNA647773 SRS7075325 GSM4688197 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688197 GSM4688197 GEO Accession GSM4688197 SRX8808951 GSM4688198 SRP273093 PRJNA647773 SRS7075327 GSM4688198 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688198 GSM4688198 GEO Accession GSM4688198 SRX8808952 GSM4688199 SRP273093 PRJNA647773 SRS7075326 GSM4688199 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688199 GSM4688199 GEO Accession GSM4688199 SRX8808953 GSM4688200 SRP273093 PRJNA647773 SRS7075328 GSM4688200 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688200 GSM4688200 GEO Accession GSM4688200 SRX8808954 GSM4688201 SRP273093 PRJNA647773 SRS7075330 GSM4688201 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688201 GSM4688201 GEO Accession GSM4688201 SRX8808955 GSM4688202 SRP273093 PRJNA647773 SRS7075329 GSM4688202 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688202 GSM4688202 GEO Accession GSM4688202 SRX8808956 GSM4688203 SRP273093 PRJNA647773 SRS7075331 GSM4688203 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688203 GSM4688203 GEO Accession GSM4688203 SRX8808957 GSM4688204 SRP273093 PRJNA647773 SRS7075332 GSM4688204 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688204 GSM4688204 GEO Accession GSM4688204 SRX8808958 GSM4688205 SRP273093 PRJNA647773 SRS7075333 GSM4688205 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688205 GSM4688205 GEO Accession GSM4688205 SRX8808959 GSM4688206 SRP273093 PRJNA647773 SRS7075336 GSM4688206 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688206 GSM4688206 GEO Accession GSM4688206 SRX8808960 GSM4688207 SRP273093 PRJNA647773 SRS7075334 GSM4688207 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688207 GSM4688207 GEO Accession GSM4688207 SRX8808961 GSM4688208 SRP273093 PRJNA647773 SRS7075335 GSM4688208 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688208 GSM4688208 GEO Accession GSM4688208 SRX8808962 GSM4688209 SRP273093 PRJNA647773 SRS7075337 GSM4688209 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688209 GSM4688209 GEO Accession GSM4688209 SRX8808963 GSM4688210 SRP273093 PRJNA647773 SRS7075338 GSM4688210 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688210 GSM4688210 GEO Accession GSM4688210 SRX8808964 GSM4688211 SRP273093 PRJNA647773 SRS7075339 GSM4688211 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688211 GSM4688211 GEO Accession GSM4688211 SRX8808965 GSM4688212 SRP273093 PRJNA647773 SRS7075341 GSM4688212 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688212 GSM4688212 GEO Accession GSM4688212 SRX8808966 GSM4688213 SRP273093 PRJNA647773 SRS7075340 GSM4688213 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688213 GSM4688213 GEO Accession GSM4688213 SRX8808967 GSM4688214 SRP273093 PRJNA647773 SRS7075343 GSM4688214 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688214 GSM4688214 GEO Accession GSM4688214 SRX8808968 GSM4688215 SRP273093 PRJNA647773 SRS7075342 GSM4688215 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688215 GSM4688215 GEO Accession GSM4688215 SRX8808969 GSM4688216 SRP273093 PRJNA647773 SRS7075344 GSM4688216 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688216 GSM4688216 GEO Accession GSM4688216 SRX8808970 GSM4688217 SRP273093 PRJNA647773 SRS7075345 GSM4688217 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688217 GSM4688217 GEO Accession GSM4688217 SRX8808971 GSM4688218 SRP273093 PRJNA647773 SRS7075346 GSM4688218 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688218 GSM4688218 GEO Accession GSM4688218 SRX8808972 GSM4688219 SRP273093 PRJNA647773 SRS7075348 GSM4688219 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688219 GSM4688219 GEO Accession GSM4688219 SRX8808973 GSM4688220 SRP273093 PRJNA647773 SRS7075347 GSM4688220 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688220 GSM4688220 GEO Accession GSM4688220 SRX8808974 GSM4688221 SRP273093 PRJNA647773 SRS7075349 GSM4688221 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688221 GSM4688221 GEO Accession GSM4688221 SRX8808975 GSM4688222 SRP273093 PRJNA647773 SRS7075351 GSM4688222 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688222 GSM4688222 GEO Accession GSM4688222 SRX8808976 GSM4688223 SRP273093 PRJNA647773 SRS7075350 GSM4688223 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688223 GSM4688223 GEO Accession GSM4688223 SRX8808977 GSM4688224 SRP273093 PRJNA647773 SRS7075352 GSM4688224 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688224 GSM4688224 GEO Accession GSM4688224 SRX8808978 GSM4688225 SRP273093 PRJNA647773 SRS7075353 GSM4688225 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688225 GSM4688225 GEO Accession GSM4688225 SRX8808979 GSM4688226 SRP273093 PRJNA647773 SRS7075354 GSM4688226 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688226 GSM4688226 GEO Accession GSM4688226 SRX8808980 GSM4688227 SRP273093 PRJNA647773 SRS7075355 GSM4688227 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688227 GSM4688227 GEO Accession GSM4688227 SRX8808981 GSM4688228 SRP273093 PRJNA647773 SRS7075357 GSM4688228 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688228 GSM4688228 GEO Accession GSM4688228 SRX8808982 GSM4688229 SRP273093 PRJNA647773 SRS7075356 GSM4688229 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688229 GSM4688229 GEO Accession GSM4688229 SRX8808983 GSM4688230 SRP273093 PRJNA647773 SRS7075358 GSM4688230 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688230 GSM4688230 GEO Accession GSM4688230 SRX8808984 GSM4688231 SRP273093 PRJNA647773 SRS7075359 GSM4688231 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688231 GSM4688231 GEO Accession GSM4688231 SRX8808985 GSM4688232 SRP273093 PRJNA647773 SRS7075360 GSM4688232 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688232 GSM4688232 GEO Accession GSM4688232 SRX8808986 GSM4688233 SRP273093 PRJNA647773 SRS7075362 GSM4688233 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688233 GSM4688233 GEO Accession GSM4688233 SRX8808987 GSM4688234 SRP273093 PRJNA647773 SRS7075361 GSM4688234 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688234 GSM4688234 GEO Accession GSM4688234 SRX8808988 GSM4688235 SRP273093 PRJNA647773 SRS7075363 GSM4688235 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688235 GSM4688235 GEO Accession GSM4688235 SRX8808989 GSM4688236 SRP273093 PRJNA647773 SRS7075364 GSM4688236 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688236 GSM4688236 GEO Accession GSM4688236 SRX8808990 GSM4688237 SRP273093 PRJNA647773 SRS7075365 GSM4688237 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688237 GSM4688237 GEO Accession GSM4688237 SRX8808991 GSM4688238 SRP273093 PRJNA647773 SRS7075367 GSM4688238 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688238 GSM4688238 GEO Accession GSM4688238 SRX8808992 GSM4688239 SRP273093 PRJNA647773 SRS7075366 GSM4688239 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688239 GSM4688239 GEO Accession GSM4688239 SRX8808993 GSM4688240 SRP273093 PRJNA647773 SRS7075368 GSM4688240 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688240 GSM4688240 GEO Accession GSM4688240 SRX8808994 GSM4688241 SRP273093 PRJNA647773 SRS7075369 GSM4688241 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688241 GSM4688241 GEO Accession GSM4688241 SRX8808995 GSM4688242 SRP273093 PRJNA647773 SRS7075370 GSM4688242 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688242 GSM4688242 GEO Accession GSM4688242 SRX8808996 GSM4688243 SRP273093 PRJNA647773 SRS7075371 GSM4688243 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688243 GSM4688243 GEO Accession GSM4688243 SRX8808997 GSM4688244 SRP273093 PRJNA647773 SRS7075373 GSM4688244 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688244 GSM4688244 GEO Accession GSM4688244 SRX8808998 GSM4688245 SRP273093 PRJNA647773 SRS7075372 GSM4688245 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688245 GSM4688245 GEO Accession GSM4688245 SRX8808999 GSM4688246 SRP273093 PRJNA647773 SRS7075375 GSM4688246 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688246 GSM4688246 GEO Accession GSM4688246 SRX8809000 GSM4688247 SRP273093 PRJNA647773 SRS7075374 GSM4688247 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688247 GSM4688247 GEO Accession GSM4688247 SRX8809001 GSM4688248 SRP273093 PRJNA647773 SRS7075376 GSM4688248 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688248 GSM4688248 GEO Accession GSM4688248 SRX8809002 GSM4688249 SRP273093 PRJNA647773 SRS7075377 GSM4688249 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688249 GSM4688249 GEO Accession GSM4688249 SRX8809003 GSM4688250 SRP273093 PRJNA647773 SRS7075378 GSM4688250 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688250 GSM4688250 GEO Accession GSM4688250 SRX8809004 GSM4688251 SRP273093 PRJNA647773 SRS7075379 GSM4688251 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688251 GSM4688251 GEO Accession GSM4688251 SRX8809005 GSM4688252 SRP273093 PRJNA647773 SRS7075380 GSM4688252 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688252 GSM4688252 GEO Accession GSM4688252 SRX8809006 GSM4688253 SRP273093 PRJNA647773 SRS7075382 GSM4688253 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688253 GSM4688253 GEO Accession GSM4688253 SRX8809007 GSM4688254 SRP273093 PRJNA647773 SRS7075381 GSM4688254 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688254 GSM4688254 GEO Accession GSM4688254 SRX8809008 GSM4688255 SRP273093 PRJNA647773 SRS7075383 GSM4688255 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688255 GSM4688255 GEO Accession GSM4688255 SRX8809009 GSM4688256 SRP273093 PRJNA647773 SRS7075384 GSM4688256 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688256 GSM4688256 GEO Accession GSM4688256 SRX8809010 GSM4688257 SRP273093 PRJNA647773 SRS7075385 GSM4688257 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688257 GSM4688257 GEO Accession GSM4688257 SRX8809011 GSM4688258 SRP273093 PRJNA647773 SRS7076145 GSM4688258 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688258 GSM4688258 GEO Accession GSM4688258 SRX8809012 GSM4688259 SRP273093 PRJNA647773 SRS7076146 GSM4688259 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688259 GSM4688259 GEO Accession GSM4688259 SRX8809013 GSM4688260 SRP273093 PRJNA647773 SRS7075386 GSM4688260 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688260 GSM4688260 GEO Accession GSM4688260 SRX8809014 GSM4688261 SRP273093 PRJNA647773 SRS7075387 GSM4688261 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688261 GSM4688261 GEO Accession GSM4688261 SRX8809015 GSM4688262 SRP273093 PRJNA647773 SRS7075390 GSM4688262 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688262 GSM4688262 GEO Accession GSM4688262 SRX8809016 GSM4688263 SRP273093 PRJNA647773 SRS7075388 GSM4688263 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688263 GSM4688263 GEO Accession GSM4688263 SRX8809017 GSM4688264 SRP273093 PRJNA647773 SRS7075389 GSM4688264 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688264 GSM4688264 GEO Accession GSM4688264 SRX8809018 GSM4688265 SRP273093 PRJNA647773 SRS7075391 GSM4688265 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688265 GSM4688265 GEO Accession GSM4688265 SRX8809019 GSM4688266 SRP273093 PRJNA647773 SRS7075392 GSM4688266 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688266 GSM4688266 GEO Accession GSM4688266 SRX8809020 GSM4688267 SRP273093 PRJNA647773 SRS7075393 GSM4688267 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688267 GSM4688267 GEO Accession GSM4688267 SRX8809021 GSM4688268 SRP273093 PRJNA647773 SRS7075394 GSM4688268 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688268 GSM4688268 GEO Accession GSM4688268 SRX8809022 GSM4688269 SRP273093 PRJNA647773 SRS7075395 GSM4688269 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688269 GSM4688269 GEO Accession GSM4688269 SRX8809023 GSM4688270 SRP273093 PRJNA647773 SRS7075396 GSM4688270 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688270 GSM4688270 GEO Accession GSM4688270 SRX8809024 GSM4688271 SRP273093 PRJNA647773 SRS7075397 GSM4688271 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688271 GSM4688271 GEO Accession GSM4688271 SRX8809025 GSM4688272 SRP273093 PRJNA647773 SRS7075398 GSM4688272 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688272 GSM4688272 GEO Accession GSM4688272 SRX8809026 GSM4688273 SRP273093 PRJNA647773 SRS7075399 GSM4688273 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688273 GSM4688273 GEO Accession GSM4688273 SRX8809027 GSM4688274 SRP273093 PRJNA647773 SRS7075400 GSM4688274 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688274 GSM4688274 GEO Accession GSM4688274 SRX8809028 GSM4688275 SRP273093 PRJNA647773 SRS7075401 GSM4688275 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688275 GSM4688275 GEO Accession GSM4688275 SRX8809029 GSM4688276 SRP273093 PRJNA647773 SRS7075404 GSM4688276 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688276 GSM4688276 GEO Accession GSM4688276 SRX8809030 GSM4688277 SRP273093 PRJNA647773 SRS7075402 GSM4688277 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688277 GSM4688277 GEO Accession GSM4688277 SRX8809031 GSM4688278 SRP273093 PRJNA647773 SRS7075407 GSM4688278 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688278 GSM4688278 GEO Accession GSM4688278 SRX8809032 GSM4688279 SRP273093 PRJNA647773 SRS7075403 GSM4688279 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688279 GSM4688279 GEO Accession GSM4688279 SRX8809033 GSM4688280 SRP273093 PRJNA647773 SRS7075405 GSM4688280 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688280 GSM4688280 GEO Accession GSM4688280 SRX8809034 GSM4688281 SRP273093 PRJNA647773 SRS7075406 GSM4688281 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688281 GSM4688281 GEO Accession GSM4688281 SRX8809035 GSM4688282 SRP273093 PRJNA647773 SRS7075408 GSM4688282 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688282 GSM4688282 GEO Accession GSM4688282 SRX8809036 GSM4688283 SRP273093 PRJNA647773 SRS7075409 GSM4688283 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688283 GSM4688283 GEO Accession GSM4688283 SRX8809037 GSM4688284 SRP273093 PRJNA647773 SRS7075410 GSM4688284 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688284 GSM4688284 GEO Accession GSM4688284 SRX8809038 GSM4688285 SRP273093 PRJNA647773 SRS7075411 GSM4688285 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688285 GSM4688285 GEO Accession GSM4688285 SRX8809039 GSM4688286 SRP273093 PRJNA647773 SRS7075412 GSM4688286 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688286 GSM4688286 GEO Accession GSM4688286 SRX8809040 GSM4688287 SRP273093 PRJNA647773 SRS7075413 GSM4688287 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688287 GSM4688287 GEO Accession GSM4688287 SRX8809041 GSM4688288 SRP273093 PRJNA647773 SRS7075414 GSM4688288 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688288 GSM4688288 GEO Accession GSM4688288 SRX8809042 GSM4688289 SRP273093 PRJNA647773 SRS7075415 GSM4688289 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688289 GSM4688289 GEO Accession GSM4688289 SRX8809043 GSM4688290 SRP273093 PRJNA647773 SRS7075416 GSM4688290 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688290 GSM4688290 GEO Accession GSM4688290 SRX8809044 GSM4688291 SRP273093 PRJNA647773 SRS7075417 GSM4688291 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688291 GSM4688291 GEO Accession GSM4688291 SRX8809045 GSM4688292 SRP273093 PRJNA647773 SRS7075418 GSM4688292 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688292 GSM4688292 GEO Accession GSM4688292 SRX8809046 GSM4688293 SRP273093 PRJNA647773 SRS7075419 GSM4688293 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688293 GSM4688293 GEO Accession GSM4688293 SRX8809047 GSM4688294 SRP273093 PRJNA647773 SRS7075420 GSM4688294 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688294 GSM4688294 GEO Accession GSM4688294 SRX8809048 GSM4688295 SRP273093 PRJNA647773 SRS7075421 GSM4688295 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688295 GSM4688295 GEO Accession GSM4688295 SRX8809049 GSM4688296 SRP273093 PRJNA647773 SRS7075422 GSM4688296 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688296 GSM4688296 GEO Accession GSM4688296 SRX8809050 GSM4688297 SRP273093 PRJNA647773 SRS7075423 GSM4688297 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688297 GSM4688297 GEO Accession GSM4688297 SRX8809051 GSM4688298 SRP273093 PRJNA647773 SRS7075424 GSM4688298 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688298 GSM4688298 GEO Accession GSM4688298 SRX8809052 GSM4688299 SRP273093 PRJNA647773 SRS7075425 GSM4688299 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688299 GSM4688299 GEO Accession GSM4688299 SRX8809053 GSM4688300 SRP273093 PRJNA647773 SRS7075426 GSM4688300 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688300 GSM4688300 GEO Accession GSM4688300 SRX8809054 GSM4688301 SRP273093 PRJNA647773 SRS7075428 GSM4688301 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688301 GSM4688301 GEO Accession GSM4688301 SRX8809055 GSM4688302 SRP273093 PRJNA647773 SRS7075427 GSM4688302 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688302 GSM4688302 GEO Accession GSM4688302 SRX8809056 GSM4688303 SRP273093 PRJNA647773 SRS7075429 GSM4688303 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688303 GSM4688303 GEO Accession GSM4688303 SRX8809057 GSM4688304 SRP273093 PRJNA647773 SRS7075430 GSM4688304 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688304 GSM4688304 GEO Accession GSM4688304 SRX8809058 GSM4688305 SRP273093 PRJNA647773 SRS7075431 GSM4688305 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688305 GSM4688305 GEO Accession GSM4688305 SRX8809059 GSM4688306 SRP273093 PRJNA647773 SRS7075432 GSM4688306 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688306 GSM4688306 GEO Accession GSM4688306 SRX8809060 GSM4688307 SRP273093 PRJNA647773 SRS7075433 GSM4688307 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688307 GSM4688307 GEO Accession GSM4688307 SRX8809061 GSM4688308 SRP273093 PRJNA647773 SRS7075434 GSM4688308 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688308 GSM4688308 GEO Accession GSM4688308 SRX8809062 GSM4688309 SRP273093 PRJNA647773 SRS7075435 GSM4688309 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688309 GSM4688309 GEO Accession GSM4688309 SRX8809063 GSM4688310 SRP273093 PRJNA647773 SRS7075436 GSM4688310 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688310 GSM4688310 GEO Accession GSM4688310 SRX8809064 GSM4688311 SRP273093 PRJNA647773 SRS7075437 GSM4688311 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688311 GSM4688311 GEO Accession GSM4688311 SRX8809065 GSM4688312 SRP273093 PRJNA647773 SRS7075438 GSM4688312 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688312 GSM4688312 GEO Accession GSM4688312 SRX8809066 GSM4688313 SRP273093 PRJNA647773 SRS7075439 GSM4688313 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688313 GSM4688313 GEO Accession GSM4688313 SRX8809067 GSM4688314 SRP273093 PRJNA647773 SRS7075440 GSM4688314 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688314 GSM4688314 GEO Accession GSM4688314 SRX8809068 GSM4688315 SRP273093 PRJNA647773 SRS7075442 GSM4688315 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688315 GSM4688315 GEO Accession GSM4688315 SRX8809069 GSM4688316 SRP273093 PRJNA647773 SRS7075441 GSM4688316 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688316 GSM4688316 GEO Accession GSM4688316 SRX8809070 GSM4688317 SRP273093 PRJNA647773 SRS7075443 GSM4688317 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688317 GSM4688317 GEO Accession GSM4688317 SRX8809071 GSM4688318 SRP273093 PRJNA647773 SRS7075445 GSM4688318 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688318 GSM4688318 GEO Accession GSM4688318 SRX8809072 GSM4688319 SRP273093 PRJNA647773 SRS7075444 GSM4688319 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688319 GSM4688319 GEO Accession GSM4688319 SRX8809073 GSM4688320 SRP273093 PRJNA647773 SRS7075446 GSM4688320 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688320 GSM4688320 GEO Accession GSM4688320 SRX8809074 GSM4688321 SRP273093 PRJNA647773 SRS7075448 GSM4688321 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688321 GSM4688321 GEO Accession GSM4688321 SRX8809075 GSM4688322 SRP273093 PRJNA647773 SRS7075447 GSM4688322 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688322 GSM4688322 GEO Accession GSM4688322 SRX8809076 GSM4688323 SRP273093 PRJNA647773 SRS7075449 GSM4688323 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688323 GSM4688323 GEO Accession GSM4688323 SRX8809077 GSM4688324 SRP273093 PRJNA647773 SRS7075450 GSM4688324 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688324 GSM4688324 GEO Accession GSM4688324 SRX8809078 GSM4688325 SRP273093 PRJNA647773 SRS7075451 GSM4688325 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688325 GSM4688325 GEO Accession GSM4688325 SRX8809079 GSM4688326 SRP273093 PRJNA647773 SRS7075452 GSM4688326 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688326 GSM4688326 GEO Accession GSM4688326 SRX8809080 GSM4688327 SRP273093 PRJNA647773 SRS7075453 GSM4688327 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688327 GSM4688327 GEO Accession GSM4688327 SRX8809081 GSM4688328 SRP273093 PRJNA647773 SRS7075454 GSM4688328 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688328 GSM4688328 GEO Accession GSM4688328 SRX8809082 GSM4688329 SRP273093 PRJNA647773 SRS7075455 GSM4688329 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688329 GSM4688329 GEO Accession GSM4688329 SRX8809083 GSM4688330 SRP273093 PRJNA647773 SRS7075456 GSM4688330 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688330 GSM4688330 GEO Accession GSM4688330 SRX8809084 GSM4688331 SRP273093 PRJNA647773 SRS7075457 GSM4688331 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688331 GSM4688331 GEO Accession GSM4688331 SRX8809085 GSM4688332 SRP273093 PRJNA647773 SRS7075458 GSM4688332 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688332 GSM4688332 GEO Accession GSM4688332 SRX8809086 GSM4688333 SRP273093 PRJNA647773 SRS7075459 GSM4688333 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688333 GSM4688333 GEO Accession GSM4688333 SRX8809087 GSM4688334 SRP273093 PRJNA647773 SRS7075460 GSM4688334 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688334 GSM4688334 GEO Accession GSM4688334 SRX8809088 GSM4688335 SRP273093 PRJNA647773 SRS7075461 GSM4688335 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688335 GSM4688335 GEO Accession GSM4688335 SRX8809089 GSM4688336 SRP273093 PRJNA647773 SRS7075462 GSM4688336 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688336 GSM4688336 GEO Accession GSM4688336 SRX8809090 GSM4688337 SRP273093 PRJNA647773 SRS7075463 GSM4688337 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688337 GSM4688337 GEO Accession GSM4688337 SRX8809091 GSM4688338 SRP273093 PRJNA647773 SRS7075464 GSM4688338 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688338 GSM4688338 GEO Accession GSM4688338 SRX8809092 GSM4688339 SRP273093 PRJNA647773 SRS7075465 GSM4688339 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688339 GSM4688339 GEO Accession GSM4688339 SRX8809093 GSM4688340 SRP273093 PRJNA647773 SRS7075466 GSM4688340 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688340 GSM4688340 GEO Accession GSM4688340 SRX8809094 GSM4688341 SRP273093 PRJNA647773 SRS7075467 GSM4688341 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688341 GSM4688341 GEO Accession GSM4688341 SRX8809095 GSM4688342 SRP273093 PRJNA647773 SRS7075468 GSM4688342 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688342 GSM4688342 GEO Accession GSM4688342 SRX8809096 GSM4688343 SRP273093 PRJNA647773 SRS7075470 GSM4688343 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688343 GSM4688343 GEO Accession GSM4688343 SRX8809097 GSM4688344 SRP273093 PRJNA647773 SRS7075469 GSM4688344 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688344 GSM4688344 GEO Accession GSM4688344 SRX8809098 GSM4688345 SRP273093 PRJNA647773 SRS7075471 GSM4688345 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688345 GSM4688345 GEO Accession GSM4688345 SRX8809099 GSM4688346 SRP273093 PRJNA647773 SRS7075472 GSM4688346 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688346 GSM4688346 GEO Accession GSM4688346 SRX8809100 GSM4688347 SRP273093 PRJNA647773 SRS7075473 GSM4688347 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688347 GSM4688347 GEO Accession GSM4688347 SRX8809101 GSM4688348 SRP273093 PRJNA647773 SRS7075475 GSM4688348 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688348 GSM4688348 GEO Accession GSM4688348 SRX8809102 GSM4688349 SRP273093 PRJNA647773 SRS7075474 GSM4688349 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688349 GSM4688349 GEO Accession GSM4688349 SRX8809103 GSM4688350 SRP273093 PRJNA647773 SRS7075477 GSM4688350 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688350 GSM4688350 GEO Accession GSM4688350 SRX8809104 GSM4688351 SRP273093 PRJNA647773 SRS7075476 GSM4688351 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688351 GSM4688351 GEO Accession GSM4688351 SRX8809105 GSM4688352 SRP273093 PRJNA647773 SRS7075478 GSM4688352 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688352 GSM4688352 GEO Accession GSM4688352 SRX8809106 GSM4688353 SRP273093 PRJNA647773 SRS7075479 GSM4688353 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688353 GSM4688353 GEO Accession GSM4688353 SRX8809107 GSM4688354 SRP273093 PRJNA647773 SRS7075480 GSM4688354 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688354 GSM4688354 GEO Accession GSM4688354 SRX8809108 GSM4688355 SRP273093 PRJNA647773 SRS7075481 GSM4688355 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688355 GSM4688355 GEO Accession GSM4688355 SRX8809109 GSM4688356 SRP273093 PRJNA647773 SRS7075482 GSM4688356 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688356 GSM4688356 GEO Accession GSM4688356 SRX8809110 GSM4688357 SRP273093 PRJNA647773 SRS7075483 GSM4688357 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688357 GSM4688357 GEO Accession GSM4688357 SRX8809111 GSM4688358 SRP273093 PRJNA647773 SRS7075484 GSM4688358 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688358 GSM4688358 GEO Accession GSM4688358 SRX8809112 GSM4688359 SRP273093 PRJNA647773 SRS7075485 GSM4688359 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688359 GSM4688359 GEO Accession GSM4688359 SRX8809113 GSM4688360 SRP273093 PRJNA647773 SRS7075486 GSM4688360 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688360 GSM4688360 GEO Accession GSM4688360 SRX8809114 GSM4688361 SRP273093 PRJNA647773 SRS7075487 GSM4688361 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688361 GSM4688361 GEO Accession GSM4688361 SRX8809115 GSM4688362 SRP273093 PRJNA647773 SRS7075488 GSM4688362 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688362 GSM4688362 GEO Accession GSM4688362 SRX8809116 GSM4688363 SRP273093 PRJNA647773 SRS7075489 GSM4688363 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688363 GSM4688363 GEO Accession GSM4688363 SRX8809117 GSM4688364 SRP273093 PRJNA647773 SRS7075490 GSM4688364 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688364 GSM4688364 GEO Accession GSM4688364 SRX8809118 GSM4688365 SRP273093 PRJNA647773 SRS7075491 GSM4688365 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688365 GSM4688365 GEO Accession GSM4688365 SRX8809119 GSM4688366 SRP273093 PRJNA647773 SRS7075492 GSM4688366 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688366 GSM4688366 GEO Accession GSM4688366 SRX8809120 GSM4688367 SRP273093 PRJNA647773 SRS7075493 GSM4688367 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688367 GSM4688367 GEO Accession GSM4688367 SRX8809121 GSM4688368 SRP273093 PRJNA647773 SRS7075494 GSM4688368 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688368 GSM4688368 GEO Accession GSM4688368 SRX8809122 GSM4688369 SRP273093 PRJNA647773 SRS7075495 GSM4688369 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688369 GSM4688369 GEO Accession GSM4688369 SRX8809123 GSM4688370 SRP273093 PRJNA647773 SRS7075496 GSM4688370 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688370 GSM4688370 GEO Accession GSM4688370 SRX8809124 GSM4688371 SRP273093 PRJNA647773 SRS7075497 GSM4688371 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688371 GSM4688371 GEO Accession GSM4688371 SRX8809125 GSM4688372 SRP273093 PRJNA647773 SRS7075498 GSM4688372 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688372 GSM4688372 GEO Accession GSM4688372 SRX8809126 GSM4688373 SRP273093 PRJNA647773 SRS7075499 GSM4688373 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688373 GSM4688373 GEO Accession GSM4688373 SRX8809127 GSM4688374 SRP273093 PRJNA647773 SRS7075502 GSM4688374 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688374 GSM4688374 GEO Accession GSM4688374 SRX8809128 GSM4688375 SRP273093 PRJNA647773 SRS7075500 GSM4688375 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688375 GSM4688375 GEO Accession GSM4688375 SRX8809129 GSM4688376 SRP273093 PRJNA647773 SRS7075501 GSM4688376 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688376 GSM4688376 GEO Accession GSM4688376 SRX8809130 GSM4688377 SRP273093 PRJNA647773 SRS7075503 GSM4688377 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688377 GSM4688377 GEO Accession GSM4688377 SRX8809131 GSM4688378 SRP273093 PRJNA647773 SRS7075505 GSM4688378 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688378 GSM4688378 GEO Accession GSM4688378 SRX8809132 GSM4688379 SRP273093 PRJNA647773 SRS7075504 GSM4688379 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688379 GSM4688379 GEO Accession GSM4688379 SRX8809133 GSM4688380 SRP273093 PRJNA647773 SRS7075506 GSM4688380 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688380 GSM4688380 GEO Accession GSM4688380 SRX8809134 GSM4688381 SRP273093 PRJNA647773 SRS7075507 GSM4688381 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688381 GSM4688381 GEO Accession GSM4688381 SRX8809135 GSM4688382 SRP273093 PRJNA647773 SRS7075508 GSM4688382 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688382 GSM4688382 GEO Accession GSM4688382 SRX8809136 GSM4688383 SRP273093 PRJNA647773 SRS7075509 GSM4688383 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688383 GSM4688383 GEO Accession GSM4688383 SRX8809137 GSM4688384 SRP273093 PRJNA647773 SRS7075510 GSM4688384 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688384 GSM4688384 GEO Accession GSM4688384 SRX8809138 GSM4688385 SRP273093 PRJNA647773 SRS7075512 GSM4688385 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688385 GSM4688385 GEO Accession GSM4688385 SRX8809139 GSM4688386 SRP273093 PRJNA647773 SRS7075511 GSM4688386 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688386 GSM4688386 GEO Accession GSM4688386 SRX8809140 GSM4688387 SRP273093 PRJNA647773 SRS7075513 GSM4688387 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688387 GSM4688387 GEO Accession GSM4688387 SRX8809141 GSM4688388 SRP273093 PRJNA647773 SRS7075514 GSM4688388 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688388 GSM4688388 GEO Accession GSM4688388 SRX8809142 GSM4688389 SRP273093 PRJNA647773 SRS7075515 GSM4688389 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688389 GSM4688389 GEO Accession GSM4688389 SRX8809143 GSM4688390 SRP273093 PRJNA647773 SRS7075516 GSM4688390 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688390 GSM4688390 GEO Accession GSM4688390 SRX8809144 GSM4688391 SRP273093 PRJNA647773 SRS7075518 GSM4688391 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688391 GSM4688391 GEO Accession GSM4688391 SRX8809145 GSM4688392 SRP273093 PRJNA647773 SRS7075517 GSM4688392 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688392 GSM4688392 GEO Accession GSM4688392 SRX8809146 GSM4688393 SRP273093 PRJNA647773 SRS7075519 GSM4688393 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688393 GSM4688393 GEO Accession GSM4688393 SRX8809147 GSM4688394 SRP273093 PRJNA647773 SRS7075520 GSM4688394 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688394 GSM4688394 GEO Accession GSM4688394 SRX8809148 GSM4688395 SRP273093 PRJNA647773 SRS7075523 GSM4688395 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688395 GSM4688395 GEO Accession GSM4688395 SRX8809149 GSM4688396 SRP273093 PRJNA647773 SRS7075521 GSM4688396 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688396 GSM4688396 GEO Accession GSM4688396 SRX8809150 GSM4688397 SRP273093 PRJNA647773 SRS7075522 GSM4688397 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688397 GSM4688397 GEO Accession GSM4688397 SRX8809151 GSM4688398 SRP273093 PRJNA647773 SRS7075524 GSM4688398 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688398 GSM4688398 GEO Accession GSM4688398 SRX8809152 GSM4688399 SRP273093 PRJNA647773 SRS7075525 GSM4688399 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688399 GSM4688399 GEO Accession GSM4688399 SRX8809153 GSM4688400 SRP273093 PRJNA647773 SRS7075526 GSM4688400 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688400 GSM4688400 GEO Accession GSM4688400 SRX8809154 GSM4688401 SRP273093 PRJNA647773 SRS7075527 GSM4688401 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688401 GSM4688401 GEO Accession GSM4688401 SRX8809155 GSM4688402 SRP273093 PRJNA647773 SRS7075528 GSM4688402 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688402 GSM4688402 GEO Accession GSM4688402 SRX8809156 GSM4688403 SRP273093 PRJNA647773 SRS7075529 GSM4688403 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688403 GSM4688403 GEO Accession GSM4688403 SRX8809157 GSM4688404 SRP273093 PRJNA647773 SRS7075530 GSM4688404 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688404 GSM4688404 GEO Accession GSM4688404 SRX8809158 GSM4688405 SRP273093 PRJNA647773 SRS7075532 GSM4688405 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688405 GSM4688405 GEO Accession GSM4688405 SRX8809159 GSM4688406 SRP273093 PRJNA647773 SRS7075531 GSM4688406 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688406 GSM4688406 GEO Accession GSM4688406 SRX8809160 GSM4688407 SRP273093 PRJNA647773 SRS7075533 GSM4688407 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688407 GSM4688407 GEO Accession GSM4688407 SRX8809161 GSM4688408 SRP273093 PRJNA647773 SRS7075534 GSM4688408 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688408 GSM4688408 GEO Accession GSM4688408 SRX8809162 GSM4688409 SRP273093 PRJNA647773 SRS7075535 GSM4688409 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688409 GSM4688409 GEO Accession GSM4688409 SRX8809163 GSM4688410 SRP273093 PRJNA647773 SRS7075537 GSM4688410 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688410 GSM4688410 GEO Accession GSM4688410 SRX8809164 GSM4688411 SRP273093 PRJNA647773 SRS7075538 GSM4688411 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688411 GSM4688411 GEO Accession GSM4688411 SRX8809165 GSM4688412 SRP273093 PRJNA647773 SRS7075539 GSM4688412 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688412 GSM4688412 GEO Accession GSM4688412 SRX8809166 GSM4688413 SRP273093 PRJNA647773 SRS7075536 GSM4688413 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688413 GSM4688413 GEO Accession GSM4688413 SRX8809167 GSM4688414 SRP273093 PRJNA647773 SRS7075540 GSM4688414 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688414 GSM4688414 GEO Accession GSM4688414 SRX8809168 GSM4688415 SRP273093 PRJNA647773 SRS7075541 GSM4688415 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688415 GSM4688415 GEO Accession GSM4688415 SRX8809169 GSM4688416 SRP273093 PRJNA647773 SRS7075542 GSM4688416 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688416 GSM4688416 GEO Accession GSM4688416 SRX8809170 GSM4688417 SRP273093 PRJNA647773 SRS7075544 GSM4688417 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688417 GSM4688417 GEO Accession GSM4688417 SRX8809171 GSM4688418 SRP273093 PRJNA647773 SRS7075543 GSM4688418 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688418 GSM4688418 GEO Accession GSM4688418 SRX8809172 GSM4688419 SRP273093 PRJNA647773 SRS7075546 GSM4688419 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688419 GSM4688419 GEO Accession GSM4688419 SRX8809173 GSM4688420 SRP273093 PRJNA647773 SRS7075545 GSM4688420 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688420 GSM4688420 GEO Accession GSM4688420 SRX8809174 GSM4688421 SRP273093 PRJNA647773 SRS7075547 GSM4688421 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688421 GSM4688421 GEO Accession GSM4688421 SRX8809175 GSM4688422 SRP273093 PRJNA647773 SRS7075548 GSM4688422 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688422 GSM4688422 GEO Accession GSM4688422 SRX8809176 GSM4688423 SRP273093 PRJNA647773 SRS7075549 GSM4688423 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688423 GSM4688423 GEO Accession GSM4688423 SRX8809177 GSM4688424 SRP273093 PRJNA647773 SRS7075551 GSM4688424 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688424 GSM4688424 GEO Accession GSM4688424 SRX8809178 GSM4688425 SRP273093 PRJNA647773 SRS7075550 GSM4688425 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688425 GSM4688425 GEO Accession GSM4688425 SRX8809179 GSM4688426 SRP273093 PRJNA647773 SRS7075553 GSM4688426 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688426 GSM4688426 GEO Accession GSM4688426 SRX8809180 GSM4688427 SRP273093 PRJNA647773 SRS7075552 GSM4688427 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688427 GSM4688427 GEO Accession GSM4688427 SRX8809181 GSM4688428 SRP273093 PRJNA647773 SRS7075554 GSM4688428 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688428 GSM4688428 GEO Accession GSM4688428 SRX8809182 GSM4688429 SRP273093 PRJNA647773 SRS7075556 GSM4688429 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688429 GSM4688429 GEO Accession GSM4688429 SRX8809183 GSM4688430 SRP273093 PRJNA647773 SRS7075555 GSM4688430 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688430 GSM4688430 GEO Accession GSM4688430 SRX8809184 GSM4688431 SRP273093 PRJNA647773 SRS7075558 GSM4688431 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688431 GSM4688431 GEO Accession GSM4688431 SRX8809185 GSM4688432 SRP273093 PRJNA647773 SRS7075557 GSM4688432 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688432 GSM4688432 GEO Accession GSM4688432 SRX8809186 GSM4688433 SRP273093 PRJNA647773 SRS7075559 GSM4688433 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688433 GSM4688433 GEO Accession GSM4688433 SRX8809187 GSM4688434 SRP273093 PRJNA647773 SRS7075561 GSM4688434 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688434 GSM4688434 GEO Accession GSM4688434 SRX8809188 GSM4688435 SRP273093 PRJNA647773 SRS7075560 GSM4688435 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688435 GSM4688435 GEO Accession GSM4688435 SRX8809189 GSM4688436 SRP273093 PRJNA647773 SRS7075562 GSM4688436 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688436 GSM4688436 GEO Accession GSM4688436 SRX8809190 GSM4688437 SRP273093 PRJNA647773 SRS7075563 GSM4688437 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688437 GSM4688437 GEO Accession GSM4688437 SRX8809191 GSM4688438 SRP273093 PRJNA647773 SRS7075565 GSM4688438 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688438 GSM4688438 GEO Accession GSM4688438 SRX8809192 GSM4688439 SRP273093 PRJNA647773 SRS7075564 GSM4688439 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688439 GSM4688439 GEO Accession GSM4688439 SRX8809193 GSM4688440 SRP273093 PRJNA647773 SRS7075566 GSM4688440 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688440 GSM4688440 GEO Accession GSM4688440 SRX8809194 GSM4688441 SRP273093 PRJNA647773 SRS7075567 GSM4688441 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688441 GSM4688441 GEO Accession GSM4688441 SRX8809195 GSM4688442 SRP273093 PRJNA647773 SRS7075568 GSM4688442 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688442 GSM4688442 GEO Accession GSM4688442 SRX8809196 GSM4688443 SRP273093 PRJNA647773 SRS7075569 GSM4688443 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688443 GSM4688443 GEO Accession GSM4688443 SRX8809197 GSM4688444 SRP273093 PRJNA647773 SRS7075570 GSM4688444 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688444 GSM4688444 GEO Accession GSM4688444 SRX8809199 GSM4688445 SRP273093 PRJNA647773 SRS7075571 GSM4688445 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688445 GSM4688445 GEO Accession GSM4688445 SRX8809200 GSM4688446 SRP273093 PRJNA647773 SRS7075573 GSM4688446 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688446 GSM4688446 GEO Accession GSM4688446 SRX8809201 GSM4688447 SRP273093 PRJNA647773 SRS7075572 GSM4688447 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688447 GSM4688447 GEO Accession GSM4688447 SRX8809202 GSM4688448 SRP273093 PRJNA647773 SRS7075574 GSM4688448 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688448 GSM4688448 GEO Accession GSM4688448 SRX8809203 GSM4688449 SRP273093 PRJNA647773 SRS7075575 GSM4688449 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688449 GSM4688449 GEO Accession GSM4688449 SRX8809204 GSM4688450 SRP273093 PRJNA647773 SRS7075577 GSM4688450 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688450 GSM4688450 GEO Accession GSM4688450 SRX8809205 GSM4688451 SRP273093 PRJNA647773 SRS7075576 GSM4688451 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688451 GSM4688451 GEO Accession GSM4688451 SRX8809206 GSM4688452 SRP273093 PRJNA647773 SRS7075578 GSM4688452 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688452 GSM4688452 GEO Accession GSM4688452 SRX8809207 GSM4688453 SRP273093 PRJNA647773 SRS7075579 GSM4688453 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688453 GSM4688453 GEO Accession GSM4688453 SRX8809208 GSM4688454 SRP273093 PRJNA647773 SRS7075580 GSM4688454 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688454 GSM4688454 GEO Accession GSM4688454 SRX8809209 GSM4688455 SRP273093 PRJNA647773 SRS7075581 GSM4688455 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688455 GSM4688455 GEO Accession GSM4688455 SRX8809210 GSM4688456 SRP273093 PRJNA647773 SRS7075583 GSM4688456 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688456 GSM4688456 GEO Accession GSM4688456 SRX8809211 GSM4688457 SRP273093 PRJNA647773 SRS7075582 GSM4688457 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688457 GSM4688457 GEO Accession GSM4688457 SRX8809212 GSM4688458 SRP273093 PRJNA647773 SRS7075585 GSM4688458 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688458 GSM4688458 GEO Accession GSM4688458 SRX8809213 GSM4688459 SRP273093 PRJNA647773 SRS7075584 GSM4688459 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688459 GSM4688459 GEO Accession GSM4688459 SRX8809214 GSM4688460 SRP273093 PRJNA647773 SRS7075587 GSM4688460 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688460 GSM4688460 GEO Accession GSM4688460 SRX8809215 GSM4688461 SRP273093 PRJNA647773 SRS7075588 GSM4688461 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688461 GSM4688461 GEO Accession GSM4688461 SRX8809216 GSM4688462 SRP273093 PRJNA647773 SRS7075586 GSM4688462 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688462 GSM4688462 GEO Accession GSM4688462 SRX8809217 GSM4688463 SRP273093 PRJNA647773 SRS7075589 GSM4688463 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688463 GSM4688463 GEO Accession GSM4688463 SRX8809218 GSM4688464 SRP273093 PRJNA647773 SRS7075590 GSM4688464 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688464 GSM4688464 GEO Accession GSM4688464 SRX8809219 GSM4688465 SRP273093 PRJNA647773 SRS7075591 GSM4688465 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688465 GSM4688465 GEO Accession GSM4688465 SRX8809220 GSM4688466 SRP273093 PRJNA647773 SRS7075593 GSM4688466 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688466 GSM4688466 GEO Accession GSM4688466 SRX8809221 GSM4688467 SRP273093 PRJNA647773 SRS7075592 GSM4688467 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688467 GSM4688467 GEO Accession GSM4688467 SRX8809222 GSM4688468 SRP273093 PRJNA647773 SRS7075594 GSM4688468 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688468 GSM4688468 GEO Accession GSM4688468 SRX8809223 GSM4688469 SRP273093 PRJNA647773 SRS7075595 GSM4688469 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688469 GSM4688469 GEO Accession GSM4688469 SRX8809224 GSM4688470 SRP273093 PRJNA647773 SRS7075596 GSM4688470 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688470 GSM4688470 GEO Accession GSM4688470 SRX8809225 GSM4688471 SRP273093 PRJNA647773 SRS7075597 GSM4688471 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688471 GSM4688471 GEO Accession GSM4688471 SRX8809226 GSM4688472 SRP273093 PRJNA647773 SRS7075599 GSM4688472 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688472 GSM4688472 GEO Accession GSM4688472 SRX8809227 GSM4688473 SRP273093 PRJNA647773 SRS7075598 GSM4688473 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688473 GSM4688473 GEO Accession GSM4688473 SRX8809228 GSM4688474 SRP273093 PRJNA647773 SRS7075600 GSM4688474 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688474 GSM4688474 GEO Accession GSM4688474 SRX8809229 GSM4688475 SRP273093 PRJNA647773 SRS7075602 GSM4688475 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688475 GSM4688475 GEO Accession GSM4688475 SRX8809230 GSM4688476 SRP273093 PRJNA647773 SRS7075601 GSM4688476 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688476 GSM4688476 GEO Accession GSM4688476 SRX8809231 GSM4688477 SRP273093 PRJNA647773 SRS7075603 GSM4688477 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688477 GSM4688477 GEO Accession GSM4688477 SRX8809232 GSM4688478 SRP273093 PRJNA647773 SRS7075605 GSM4688478 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688478 GSM4688478 GEO Accession GSM4688478 SRX8809233 GSM4688479 SRP273093 PRJNA647773 SRS7075604 GSM4688479 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688479 GSM4688479 GEO Accession GSM4688479 SRX8809234 GSM4688480 SRP273093 PRJNA647773 SRS7075606 GSM4688480 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688480 GSM4688480 GEO Accession GSM4688480 SRX8809235 GSM4688481 SRP273093 PRJNA647773 SRS7075607 GSM4688481 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688481 GSM4688481 GEO Accession GSM4688481 SRX8809236 GSM4688482 SRP273093 PRJNA647773 SRS7075609 GSM4688482 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688482 GSM4688482 GEO Accession GSM4688482 SRX8809237 GSM4688483 SRP273093 PRJNA647773 SRS7075608 GSM4688483 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688483 GSM4688483 GEO Accession GSM4688483 SRX8809238 GSM4688484 SRP273093 PRJNA647773 SRS7075610 GSM4688484 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688484 GSM4688484 GEO Accession GSM4688484 SRX8809239 GSM4688485 SRP273093 PRJNA647773 SRS7075611 GSM4688485 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688485 GSM4688485 GEO Accession GSM4688485 SRX8809241 GSM4688486 SRP273093 PRJNA647773 SRS7075612 GSM4688486 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688486 GSM4688486 GEO Accession GSM4688486 SRX8809242 GSM4688487 SRP273093 PRJNA647773 SRS7075613 GSM4688487 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688487 GSM4688487 GEO Accession GSM4688487 SRX8809243 GSM4688488 SRP273093 PRJNA647773 SRS7075614 GSM4688488 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688488 GSM4688488 GEO Accession GSM4688488 SRX8809244 GSM4688489 SRP273093 PRJNA647773 SRS7075615 GSM4688489 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688489 GSM4688489 GEO Accession GSM4688489 SRX8809245 GSM4688490 SRP273093 PRJNA647773 SRS7075616 GSM4688490 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688490 GSM4688490 GEO Accession GSM4688490 SRX8809246 GSM4688491 SRP273093 PRJNA647773 SRS7075617 GSM4688491 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688491 GSM4688491 GEO Accession GSM4688491 SRX8809247 GSM4688492 SRP273093 PRJNA647773 SRS7075618 GSM4688492 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688492 GSM4688492 GEO Accession GSM4688492 SRX8809248 GSM4688493 SRP273093 PRJNA647773 SRS7075619 GSM4688493 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688493 GSM4688493 GEO Accession GSM4688493 SRX8809249 GSM4688494 SRP273093 PRJNA647773 SRS7075620 GSM4688494 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688494 GSM4688494 GEO Accession GSM4688494 SRX8809250 GSM4688495 SRP273093 PRJNA647773 SRS7075621 GSM4688495 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688495 GSM4688495 GEO Accession GSM4688495 SRX8809251 GSM4688496 SRP273093 PRJNA647773 SRS7076148 GSM4688496 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688496 GSM4688496 GEO Accession GSM4688496 SRX8809252 GSM4688497 SRP273093 PRJNA647773 SRS7076147 GSM4688497 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688497 GSM4688497 GEO Accession GSM4688497 SRX8809253 GSM4688498 SRP273093 PRJNA647773 SRS7076150 GSM4688498 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688498 GSM4688498 GEO Accession GSM4688498 SRX8809254 GSM4688499 SRP273093 PRJNA647773 SRS7076149 GSM4688499 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688499 GSM4688499 GEO Accession GSM4688499 SRX8809255 GSM4688500 SRP273093 PRJNA647773 SRS7076151 GSM4688500 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688500 GSM4688500 GEO Accession GSM4688500 SRX8809256 GSM4688501 SRP273093 PRJNA647773 SRS7076153 GSM4688501 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688501 GSM4688501 GEO Accession GSM4688501 SRX8809257 GSM4688502 SRP273093 PRJNA647773 SRS7076152 GSM4688502 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688502 GSM4688502 GEO Accession GSM4688502 SRX8809258 GSM4688503 SRP273093 PRJNA647773 SRS7076154 GSM4688503 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688503 GSM4688503 GEO Accession GSM4688503 SRX8809259 GSM4688504 SRP273093 PRJNA647773 SRS7076155 GSM4688504 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688504 GSM4688504 GEO Accession GSM4688504 SRX8809260 GSM4688505 SRP273093 PRJNA647773 SRS7076158 GSM4688505 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688505 GSM4688505 GEO Accession GSM4688505 SRX8809261 GSM4688506 SRP273093 PRJNA647773 SRS7076156 GSM4688506 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688506 GSM4688506 GEO Accession GSM4688506 SRX8809262 GSM4688507 SRP273093 PRJNA647773 SRS7076157 GSM4688507 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688507 GSM4688507 GEO Accession GSM4688507 SRX8809263 GSM4688508 SRP273093 PRJNA647773 SRS7076159 GSM4688508 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688508 GSM4688508 GEO Accession GSM4688508 SRX8809264 GSM4688509 SRP273093 PRJNA647773 SRS7076161 GSM4688509 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688509 GSM4688509 GEO Accession GSM4688509 SRX8809265 GSM4688510 SRP273093 PRJNA647773 SRS7076160 GSM4688510 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688510 GSM4688510 GEO Accession GSM4688510 SRX8809266 GSM4688511 SRP273093 PRJNA647773 SRS7076162 GSM4688511 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688511 GSM4688511 GEO Accession GSM4688511 SRX8809267 GSM4688512 SRP273093 PRJNA647773 SRS7076163 GSM4688512 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688512 GSM4688512 GEO Accession GSM4688512 SRX8809268 GSM4688513 SRP273093 PRJNA647773 SRS7076164 GSM4688513 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688513 GSM4688513 GEO Accession GSM4688513 SRX8809269 GSM4688514 SRP273093 PRJNA647773 SRS7076166 GSM4688514 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688514 GSM4688514 GEO Accession GSM4688514 SRX8809270 GSM4688515 SRP273093 PRJNA647773 SRS7076165 GSM4688515 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688515 GSM4688515 GEO Accession GSM4688515 SRX8809271 GSM4688516 SRP273093 PRJNA647773 SRS7076168 GSM4688516 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688516 GSM4688516 GEO Accession GSM4688516 SRX8809272 GSM4688517 SRP273093 PRJNA647773 SRS7076167 GSM4688517 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688517 GSM4688517 GEO Accession GSM4688517 SRX8809273 GSM4688518 SRP273093 PRJNA647773 SRS7076170 GSM4688518 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688518 GSM4688518 GEO Accession GSM4688518 SRX8809274 GSM4688519 SRP273093 PRJNA647773 SRS7076169 GSM4688519 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688519 GSM4688519 GEO Accession GSM4688519 SRX8809275 GSM4688520 SRP273093 PRJNA647773 SRS7076171 GSM4688520 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688520 GSM4688520 GEO Accession GSM4688520 SRX8809276 GSM4688521 SRP273093 PRJNA647773 SRS7076172 GSM4688521 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688521 GSM4688521 GEO Accession GSM4688521 SRX8809277 GSM4688522 SRP273093 PRJNA647773 SRS7076175 GSM4688522 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688522 GSM4688522 GEO Accession GSM4688522 SRX8809278 GSM4688523 SRP273093 PRJNA647773 SRS7076173 GSM4688523 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688523 GSM4688523 GEO Accession GSM4688523 SRX8809279 GSM4688524 SRP273093 PRJNA647773 SRS7076174 GSM4688524 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688524 GSM4688524 GEO Accession GSM4688524 SRX8809281 GSM4688525 SRP273093 PRJNA647773 SRS7076176 GSM4688525 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688525 GSM4688525 GEO Accession GSM4688525 SRX8809282 GSM4688526 SRP273093 PRJNA647773 SRS7076178 GSM4688526 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688526 GSM4688526 GEO Accession GSM4688526 SRX8809283 GSM4688527 SRP273093 PRJNA647773 SRS7076177 GSM4688527 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688527 GSM4688527 GEO Accession GSM4688527 SRX8809284 GSM4688528 SRP273093 PRJNA647773 SRS7076179 GSM4688528 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688528 GSM4688528 GEO Accession GSM4688528 SRX8809285 GSM4688529 SRP273093 PRJNA647773 SRS7076180 GSM4688529 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688529 GSM4688529 GEO Accession GSM4688529 SRX8809286 GSM4688530 SRP273093 PRJNA647773 SRS7076181 GSM4688530 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688530 GSM4688530 GEO Accession GSM4688530 SRX8809287 GSM4688531 SRP273093 PRJNA647773 SRS7076183 GSM4688531 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688531 GSM4688531 GEO Accession GSM4688531 SRX8809288 GSM4688532 SRP273093 PRJNA647773 SRS7076187 GSM4688532 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688532 GSM4688532 GEO Accession GSM4688532 SRX8809289 GSM4688533 SRP273093 PRJNA647773 SRS7076182 GSM4688533 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688533 GSM4688533 GEO Accession GSM4688533 SRX8809290 GSM4688534 SRP273093 PRJNA647773 SRS7076185 GSM4688534 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688534 GSM4688534 GEO Accession GSM4688534 SRX8809292 GSM4688536 SRP273093 PRJNA647773 SRS7076186 GSM4688536 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688536 GSM4688536 GEO Accession GSM4688536 SRX8809293 GSM4688537 SRP273093 PRJNA647773 SRS7076188 GSM4688537 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688537 GSM4688537 GEO Accession GSM4688537 SRX8809294 GSM4688538 SRP273093 PRJNA647773 SRS7076193 GSM4688538 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688538 GSM4688538 GEO Accession GSM4688538 SRX8809295 GSM4688539 SRP273093 PRJNA647773 SRS7076189 GSM4688539 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688539 GSM4688539 GEO Accession GSM4688539 SRX8809296 GSM4688540 SRP273093 PRJNA647773 SRS7076190 GSM4688540 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688540 GSM4688540 GEO Accession GSM4688540 SRX8809297 GSM4688541 SRP273093 PRJNA647773 SRS7076191 GSM4688541 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688541 GSM4688541 GEO Accession GSM4688541 SRX8809298 GSM4688542 SRP273093 PRJNA647773 SRS7076192 GSM4688542 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688542 GSM4688542 GEO Accession GSM4688542 SRX8809299 GSM4688543 SRP273093 PRJNA647773 SRS7076194 GSM4688543 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688543 GSM4688543 GEO Accession GSM4688543 SRX8809300 GSM4688544 SRP273093 PRJNA647773 SRS7076195 GSM4688544 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688544 GSM4688544 GEO Accession GSM4688544 SRX8809301 GSM4688545 SRP273093 PRJNA647773 SRS7076198 GSM4688545 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688545 GSM4688545 GEO Accession GSM4688545 SRX8809302 GSM4688546 SRP273093 PRJNA647773 SRS7076196 GSM4688546 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688546 GSM4688546 GEO Accession GSM4688546 SRX8809303 GSM4688547 SRP273093 PRJNA647773 SRS7076197 GSM4688547 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688547 GSM4688547 GEO Accession GSM4688547 SRX8809304 GSM4688548 SRP273093 PRJNA647773 SRS7076199 GSM4688548 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688548 GSM4688548 GEO Accession GSM4688548 SRX8809305 GSM4688549 SRP273093 PRJNA647773 SRS7076201 GSM4688549 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688549 GSM4688549 GEO Accession GSM4688549 SRX8809306 GSM4688550 SRP273093 PRJNA647773 SRS7076200 GSM4688550 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688550 GSM4688550 GEO Accession GSM4688550 SRX8809307 GSM4688551 SRP273093 PRJNA647773 SRS7076204 GSM4688551 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688551 GSM4688551 GEO Accession GSM4688551 SRX8809308 GSM4688552 SRP273093 PRJNA647773 SRS7076202 GSM4688552 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688552 GSM4688552 GEO Accession GSM4688552 SRX8809309 GSM4688553 SRP273093 PRJNA647773 SRS7076203 GSM4688553 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688553 GSM4688553 GEO Accession GSM4688553 SRX8809310 GSM4688554 SRP273093 PRJNA647773 SRS7076205 GSM4688554 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688554 GSM4688554 GEO Accession GSM4688554 SRX8809311 GSM4688555 SRP273093 PRJNA647773 SRS7076206 GSM4688555 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688555 GSM4688555 GEO Accession GSM4688555 SRX8809312 GSM4688556 SRP273093 PRJNA647773 SRS7076207 GSM4688556 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688556 GSM4688556 GEO Accession GSM4688556 SRX8809313 GSM4688557 SRP273093 PRJNA647773 SRS7076209 GSM4688557 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688557 GSM4688557 GEO Accession GSM4688557 SRX8809315 GSM4688558 SRP273093 PRJNA647773 SRS7076208 GSM4688558 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688558 GSM4688558 GEO Accession GSM4688558 SRX8809316 GSM4688559 SRP273093 PRJNA647773 SRS7076210 GSM4688559 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688559 GSM4688559 GEO Accession GSM4688559 SRX8809317 GSM4688560 SRP273093 PRJNA647773 SRS7076213 GSM4688560 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688560 GSM4688560 GEO Accession GSM4688560 SRX8809318 GSM4688561 SRP273093 PRJNA647773 SRS7076211 GSM4688561 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688561 GSM4688561 GEO Accession GSM4688561 SRX8809319 GSM4688562 SRP273093 PRJNA647773 SRS7076214 GSM4688562 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688562 GSM4688562 GEO Accession GSM4688562 SRX8809320 GSM4688563 SRP273093 PRJNA647773 SRS7076212 GSM4688563 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688563 GSM4688563 GEO Accession GSM4688563 SRX8809321 GSM4688564 SRP273093 PRJNA647773 SRS7076216 GSM4688564 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688564 GSM4688564 GEO Accession GSM4688564 SRX8809322 GSM4688565 SRP273093 PRJNA647773 SRS7076215 GSM4688565 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688565 GSM4688565 GEO Accession GSM4688565 SRX8809323 GSM4688566 SRP273093 PRJNA647773 SRS7076217 GSM4688566 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688566 GSM4688566 GEO Accession GSM4688566 SRX8809324 GSM4688567 SRP273093 PRJNA647773 SRS7076218 GSM4688567 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688567 GSM4688567 GEO Accession GSM4688567 SRX8809938 GSM4689181 SRP273093 PRJNA647773 SRS7077108 GSM4689181 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689181 GSM4689181 GEO Accession GSM4689181 SRX8809939 GSM4689182 SRP273093 PRJNA647773 SRS7077107 GSM4689182 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689182 GSM4689182 GEO Accession GSM4689182 SRX8809940 GSM4689183 SRP273093 PRJNA647773 SRS7077110 GSM4689183 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689183 GSM4689183 GEO Accession GSM4689183 SRX8809941 GSM4689184 SRP273093 PRJNA647773 SRS7077109 GSM4689184 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689184 GSM4689184 GEO Accession GSM4689184 SRX8809942 GSM4689185 SRP273093 PRJNA647773 SRS7077111 GSM4689185 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689185 GSM4689185 GEO Accession GSM4689185 SRX8809943 GSM4689186 SRP273093 PRJNA647773 SRS7077112 GSM4689186 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689186 GSM4689186 GEO Accession GSM4689186 SRX8809944 GSM4689187 SRP273093 PRJNA647773 SRS7077114 GSM4689187 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689187 GSM4689187 GEO Accession GSM4689187 SRX8809945 GSM4689188 SRP273093 PRJNA647773 SRS7077113 GSM4689188 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689188 GSM4689188 GEO Accession GSM4689188 SRX8809946 GSM4689189 SRP273093 PRJNA647773 SRS7077115 GSM4689189 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689189 GSM4689189 GEO Accession GSM4689189 SRX8809947 GSM4689190 SRP273093 PRJNA647773 SRS7077117 GSM4689190 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689190 GSM4689190 GEO Accession GSM4689190 SRX8809948 GSM4689191 SRP273093 PRJNA647773 SRS7077118 GSM4689191 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689191 GSM4689191 GEO Accession GSM4689191 SRX8809949 GSM4689192 SRP273093 PRJNA647773 SRS7077116 GSM4689192 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689192 GSM4689192 GEO Accession GSM4689192 SRX8809950 GSM4689193 SRP273093 PRJNA647773 SRS7077119 GSM4689193 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689193 GSM4689193 GEO Accession GSM4689193 SRX8809951 GSM4689194 SRP273093 PRJNA647773 SRS7077120 GSM4689194 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689194 GSM4689194 GEO Accession GSM4689194 SRX8809952 GSM4689195 SRP273093 PRJNA647773 SRS7077121 GSM4689195 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689195 GSM4689195 GEO Accession GSM4689195 SRX8809953 GSM4689196 SRP273093 PRJNA647773 SRS7077122 GSM4689196 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689196 GSM4689196 GEO Accession GSM4689196 SRX8809954 GSM4689197 SRP273093 PRJNA647773 SRS7077123 GSM4689197 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689197 GSM4689197 GEO Accession GSM4689197 SRX8809955 GSM4689198 SRP273093 PRJNA647773 SRS7077125 GSM4689198 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689198 GSM4689198 GEO Accession GSM4689198 SRX8809956 GSM4689199 SRP273093 PRJNA647773 SRS7077124 GSM4689199 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689199 GSM4689199 GEO Accession GSM4689199 SRX8809957 GSM4689200 SRP273093 PRJNA647773 SRS7077126 GSM4689200 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689200 GSM4689200 GEO Accession GSM4689200 SRX8809958 GSM4689201 SRP273093 PRJNA647773 SRS7077127 GSM4689201 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689201 GSM4689201 GEO Accession GSM4689201 SRX8809959 GSM4689202 SRP273093 PRJNA647773 SRS7077128 GSM4689202 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689202 GSM4689202 GEO Accession GSM4689202 SRX8809960 GSM4689203 SRP273093 PRJNA647773 SRS7077130 GSM4689203 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689203 GSM4689203 GEO Accession GSM4689203 SRX8809961 GSM4689204 SRP273093 PRJNA647773 SRS7077129 GSM4689204 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689204 GSM4689204 GEO Accession GSM4689204 SRX8809962 GSM4689205 SRP273093 PRJNA647773 SRS7077132 GSM4689205 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689205 GSM4689205 GEO Accession GSM4689205 SRX8809963 GSM4689206 SRP273093 PRJNA647773 SRS7077131 GSM4689206 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689206 GSM4689206 GEO Accession GSM4689206 SRX8809964 GSM4689207 SRP273093 PRJNA647773 SRS7077134 GSM4689207 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689207 GSM4689207 GEO Accession GSM4689207 SRX8809965 GSM4689208 SRP273093 PRJNA647773 SRS7077133 GSM4689208 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689208 GSM4689208 GEO Accession GSM4689208 SRX8809966 GSM4689209 SRP273093 PRJNA647773 SRS7077135 GSM4689209 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689209 GSM4689209 GEO Accession GSM4689209 SRX8809967 GSM4689210 SRP273093 PRJNA647773 SRS7077136 GSM4689210 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689210 GSM4689210 GEO Accession GSM4689210 SRX8809968 GSM4689211 SRP273093 PRJNA647773 SRS7077138 GSM4689211 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689211 GSM4689211 GEO Accession GSM4689211 SRX8809969 GSM4689212 SRP273093 PRJNA647773 SRS7077137 GSM4689212 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689212 GSM4689212 GEO Accession GSM4689212 SRX8809970 GSM4689213 SRP273093 PRJNA647773 SRS7077139 GSM4689213 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689213 GSM4689213 GEO Accession GSM4689213 SRX8809971 GSM4689214 SRP273093 PRJNA647773 SRS7077140 GSM4689214 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689214 GSM4689214 GEO Accession GSM4689214 SRX8809972 GSM4689215 SRP273093 PRJNA647773 SRS7077141 GSM4689215 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689215 GSM4689215 GEO Accession GSM4689215 SRX8809973 GSM4689216 SRP273093 PRJNA647773 SRS7077144 GSM4689216 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689216 GSM4689216 GEO Accession GSM4689216 SRX8809974 GSM4689217 SRP273093 PRJNA647773 SRS7077142 GSM4689217 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689217 GSM4689217 GEO Accession GSM4689217 SRX8809975 GSM4689218 SRP273093 PRJNA647773 SRS7077143 GSM4689218 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689218 GSM4689218 GEO Accession GSM4689218 SRX8809976 GSM4689219 SRP273093 PRJNA647773 SRS7077146 GSM4689219 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689219 GSM4689219 GEO Accession GSM4689219 SRX8809977 GSM4689220 SRP273093 PRJNA647773 SRS7077145 GSM4689220 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689220 GSM4689220 GEO Accession GSM4689220 SRX8809978 GSM4689221 SRP273093 PRJNA647773 SRS7077150 GSM4689221 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689221 GSM4689221 GEO Accession GSM4689221 SRX8809979 GSM4689222 SRP273093 PRJNA647773 SRS7077147 GSM4689222 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689222 GSM4689222 GEO Accession GSM4689222 SRX8809980 GSM4689223 SRP273093 PRJNA647773 SRS7077148 GSM4689223 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689223 GSM4689223 GEO Accession GSM4689223 SRX8809981 GSM4689224 SRP273093 PRJNA647773 SRS7077149 GSM4689224 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689224 GSM4689224 GEO Accession GSM4689224 SRX8809982 GSM4689225 SRP273093 PRJNA647773 SRS7077151 GSM4689225 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689225 GSM4689225 GEO Accession GSM4689225 SRX8809983 GSM4689226 SRP273093 PRJNA647773 SRS7077152 GSM4689226 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689226 GSM4689226 GEO Accession GSM4689226 SRX8809984 GSM4689227 SRP273093 PRJNA647773 SRS7077153 GSM4689227 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689227 GSM4689227 GEO Accession GSM4689227 SRX8809985 GSM4689228 SRP273093 PRJNA647773 SRS7077154 GSM4689228 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689228 GSM4689228 GEO Accession GSM4689228 SRX8809986 GSM4689229 SRP273093 PRJNA647773 SRS7077155 GSM4689229 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689229 GSM4689229 GEO Accession GSM4689229 SRX8809987 GSM4689230 SRP273093 PRJNA647773 SRS7077156 GSM4689230 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689230 GSM4689230 GEO Accession GSM4689230 SRX8809988 GSM4689231 SRP273093 PRJNA647773 SRS7077159 GSM4689231 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689231 GSM4689231 GEO Accession GSM4689231 SRX8809989 GSM4689232 SRP273093 PRJNA647773 SRS7077157 GSM4689232 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689232 GSM4689232 GEO Accession GSM4689232 SRX8809990 GSM4689233 SRP273093 PRJNA647773 SRS7077160 GSM4689233 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689233 GSM4689233 GEO Accession GSM4689233 SRX8809991 GSM4689234 SRP273093 PRJNA647773 SRS7077158 GSM4689234 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689234 GSM4689234 GEO Accession GSM4689234 SRX8809992 GSM4689235 SRP273093 PRJNA647773 SRS7077161 GSM4689235 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689235 GSM4689235 GEO Accession GSM4689235 SRX8809993 GSM4689236 SRP273093 PRJNA647773 SRS7077162 GSM4689236 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689236 GSM4689236 GEO Accession GSM4689236 SRX8809994 GSM4689237 SRP273093 PRJNA647773 SRS7077164 GSM4689237 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689237 GSM4689237 GEO Accession GSM4689237 SRX8809995 GSM4689238 SRP273093 PRJNA647773 SRS7077163 GSM4689238 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689238 GSM4689238 GEO Accession GSM4689238 SRX8809996 GSM4689239 SRP273093 PRJNA647773 SRS7077165 GSM4689239 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689239 GSM4689239 GEO Accession GSM4689239 SRX8809997 GSM4689240 SRP273093 PRJNA647773 SRS7076219 GSM4689240 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689240 GSM4689240 GEO Accession GSM4689240 SRX8809998 GSM4689241 SRP273093 PRJNA647773 SRS7076220 GSM4689241 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689241 GSM4689241 GEO Accession GSM4689241 SRX8809999 GSM4689242 SRP273093 PRJNA647773 SRS7076221 GSM4689242 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689242 GSM4689242 GEO Accession GSM4689242 SRX8810000 GSM4689243 SRP273093 PRJNA647773 SRS7076222 GSM4689243 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689243 GSM4689243 GEO Accession GSM4689243 SRX8810001 GSM4689244 SRP273093 PRJNA647773 SRS7077166 GSM4689244 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689244 GSM4689244 GEO Accession GSM4689244 SRX8810002 GSM4689245 SRP273093 PRJNA647773 SRS7077167 GSM4689245 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689245 GSM4689245 GEO Accession GSM4689245 SRX8810003 GSM4689246 SRP273093 PRJNA647773 SRS7077168 GSM4689246 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689246 GSM4689246 GEO Accession GSM4689246 SRX8810004 GSM4689247 SRP273093 PRJNA647773 SRS7077169 GSM4689247 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689247 GSM4689247 GEO Accession GSM4689247 SRX8810005 GSM4689248 SRP273093 PRJNA647773 SRS7077170 GSM4689248 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689248 GSM4689248 GEO Accession GSM4689248 SRX8810006 GSM4689249 SRP273093 PRJNA647773 SRS7077172 GSM4689249 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689249 GSM4689249 GEO Accession GSM4689249 SRX8810007 GSM4689250 SRP273093 PRJNA647773 SRS7077171 GSM4689250 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689250 GSM4689250 GEO Accession GSM4689250 SRX8810008 GSM4689251 SRP273093 PRJNA647773 SRS7077173 GSM4689251 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689251 GSM4689251 GEO Accession GSM4689251 SRX8810009 GSM4689252 SRP273093 PRJNA647773 SRS7077175 GSM4689252 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689252 GSM4689252 GEO Accession GSM4689252 SRX8810010 GSM4689253 SRP273093 PRJNA647773 SRS7077174 GSM4689253 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689253 GSM4689253 GEO Accession GSM4689253 SRX8810011 GSM4689254 SRP273093 PRJNA647773 SRS7077177 GSM4689254 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689254 GSM4689254 GEO Accession GSM4689254 SRX8810012 GSM4689255 SRP273093 PRJNA647773 SRS7077176 GSM4689255 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689255 GSM4689255 GEO Accession GSM4689255 SRX8810013 GSM4689256 SRP273093 PRJNA647773 SRS7077178 GSM4689256 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689256 GSM4689256 GEO Accession GSM4689256 SRX8810014 GSM4689257 SRP273093 PRJNA647773 SRS7077179 GSM4689257 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689257 GSM4689257 GEO Accession GSM4689257 SRX8810015 GSM4689258 SRP273093 PRJNA647773 SRS7077180 GSM4689258 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689258 GSM4689258 GEO Accession GSM4689258 SRX8810016 GSM4689259 SRP273093 PRJNA647773 SRS7077182 GSM4689259 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689259 GSM4689259 GEO Accession GSM4689259 SRX8810017 GSM4689260 SRP273093 PRJNA647773 SRS7077181 GSM4689260 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689260 GSM4689260 GEO Accession GSM4689260 SRX8810018 GSM4689261 SRP273093 PRJNA647773 SRS7077183 GSM4689261 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689261 GSM4689261 GEO Accession GSM4689261 SRX8810019 GSM4689262 SRP273093 PRJNA647773 SRS7077184 GSM4689262 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689262 GSM4689262 GEO Accession GSM4689262 SRX8810020 GSM4689263 SRP273093 PRJNA647773 SRS7077185 GSM4689263 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689263 GSM4689263 GEO Accession GSM4689263 SRX8810021 GSM4689264 SRP273093 PRJNA647773 SRS7077186 GSM4689264 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689264 GSM4689264 GEO Accession GSM4689264 SRX8810022 GSM4689265 SRP273093 PRJNA647773 SRS7077188 GSM4689265 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689265 GSM4689265 GEO Accession GSM4689265 SRX8810023 GSM4689266 SRP273093 PRJNA647773 SRS7077187 GSM4689266 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689266 GSM4689266 GEO Accession GSM4689266 SRX8810024 GSM4689267 SRP273093 PRJNA647773 SRS7077189 GSM4689267 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689267 GSM4689267 GEO Accession GSM4689267 SRX8810025 GSM4689268 SRP273093 PRJNA647773 SRS7077190 GSM4689268 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689268 GSM4689268 GEO Accession GSM4689268 SRX8810026 GSM4689269 SRP273093 PRJNA647773 SRS7077192 GSM4689269 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689269 GSM4689269 GEO Accession GSM4689269 SRX8810027 GSM4689270 SRP273093 PRJNA647773 SRS7077191 GSM4689270 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689270 GSM4689270 GEO Accession GSM4689270 SRX8810028 GSM4689271 SRP273093 PRJNA647773 SRS7077193 GSM4689271 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689271 GSM4689271 GEO Accession GSM4689271 SRX8810029 GSM4689272 SRP273093 PRJNA647773 SRS7077194 GSM4689272 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689272 GSM4689272 GEO Accession GSM4689272 SRX8810030 GSM4689273 SRP273093 PRJNA647773 SRS7077196 GSM4689273 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689273 GSM4689273 GEO Accession GSM4689273 SRX8810031 GSM4689274 SRP273093 PRJNA647773 SRS7077195 GSM4689274 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689274 GSM4689274 GEO Accession GSM4689274 SRX8810032 GSM4689275 SRP273093 PRJNA647773 SRS7077197 GSM4689275 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689275 GSM4689275 GEO Accession GSM4689275 SRX8810033 GSM4689276 SRP273093 PRJNA647773 SRS7077198 GSM4689276 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689276 GSM4689276 GEO Accession GSM4689276 SRX8810034 GSM4689277 SRP273093 PRJNA647773 SRS7077199 GSM4689277 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689277 GSM4689277 GEO Accession GSM4689277 SRX8810035 GSM4689278 SRP273093 PRJNA647773 SRS7077201 GSM4689278 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689278 GSM4689278 GEO Accession GSM4689278 SRX8810036 GSM4689279 SRP273093 PRJNA647773 SRS7077200 GSM4689279 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689279 GSM4689279 GEO Accession GSM4689279 SRX8810037 GSM4689280 SRP273093 PRJNA647773 SRS7077203 GSM4689280 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689280 GSM4689280 GEO Accession GSM4689280 SRX8810038 GSM4689281 SRP273093 PRJNA647773 SRS7077202 GSM4689281 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689281 GSM4689281 GEO Accession GSM4689281 SRX8810039 GSM4689282 SRP273093 PRJNA647773 SRS7077204 GSM4689282 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689282 GSM4689282 GEO Accession GSM4689282 SRX8810040 GSM4689283 SRP273093 PRJNA647773 SRS7077207 GSM4689283 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689283 GSM4689283 GEO Accession GSM4689283 SRX8810041 GSM4689284 SRP273093 PRJNA647773 SRS7077205 GSM4689284 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689284 GSM4689284 GEO Accession GSM4689284 SRX8810042 GSM4689285 SRP273093 PRJNA647773 SRS7077206 GSM4689285 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689285 GSM4689285 GEO Accession GSM4689285 SRX8810043 GSM4689286 SRP273093 PRJNA647773 SRS7077208 GSM4689286 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689286 GSM4689286 GEO Accession GSM4689286 SRX8810044 GSM4689287 SRP273093 PRJNA647773 SRS7077211 GSM4689287 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689287 GSM4689287 GEO Accession GSM4689287 SRX8810045 GSM4689288 SRP273093 PRJNA647773 SRS7077210 GSM4689288 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689288 GSM4689288 GEO Accession GSM4689288 SRX8810046 GSM4689289 SRP273093 PRJNA647773 SRS7077212 GSM4689289 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689289 GSM4689289 GEO Accession GSM4689289 SRX8810047 GSM4689290 SRP273093 PRJNA647773 SRS7077209 GSM4689290 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689290 GSM4689290 GEO Accession GSM4689290 SRX8810048 GSM4689291 SRP273093 PRJNA647773 SRS7077214 GSM4689291 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689291 GSM4689291 GEO Accession GSM4689291 SRX8810049 GSM4689292 SRP273093 PRJNA647773 SRS7077213 GSM4689292 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689292 GSM4689292 GEO Accession GSM4689292 SRX8810050 GSM4689293 SRP273093 PRJNA647773 SRS7077217 GSM4689293 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689293 GSM4689293 GEO Accession GSM4689293 SRX8810051 GSM4689294 SRP273093 PRJNA647773 SRS7077215 GSM4689294 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689294 GSM4689294 GEO Accession GSM4689294 SRX8810052 GSM4689295 SRP273093 PRJNA647773 SRS7077216 GSM4689295 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689295 GSM4689295 GEO Accession GSM4689295 SRX8810053 GSM4689296 SRP273093 PRJNA647773 SRS7077218 GSM4689296 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689296 GSM4689296 GEO Accession GSM4689296 SRX8810054 GSM4689297 SRP273093 PRJNA647773 SRS7077220 GSM4689297 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689297 GSM4689297 GEO Accession GSM4689297 SRX8810055 GSM4689298 SRP273093 PRJNA647773 SRS7077219 GSM4689298 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689298 GSM4689298 GEO Accession GSM4689298 SRX8810056 GSM4689299 SRP273093 PRJNA647773 SRS7077221 GSM4689299 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689299 GSM4689299 GEO Accession GSM4689299 SRX8810057 GSM4689300 SRP273093 PRJNA647773 SRS7077222 GSM4689300 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689300 GSM4689300 GEO Accession GSM4689300 SRX8810058 GSM4689301 SRP273093 PRJNA647773 SRS7077223 GSM4689301 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689301 GSM4689301 GEO Accession GSM4689301 SRX8810059 GSM4689302 SRP273093 PRJNA647773 SRS7077226 GSM4689302 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689302 GSM4689302 GEO Accession GSM4689302 SRX8810060 GSM4689303 SRP273093 PRJNA647773 SRS7077224 GSM4689303 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689303 GSM4689303 GEO Accession GSM4689303 SRX8810061 GSM4689304 SRP273093 PRJNA647773 SRS7077225 GSM4689304 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689304 GSM4689304 GEO Accession GSM4689304 SRX8810062 GSM4689305 SRP273093 PRJNA647773 SRS7077227 GSM4689305 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689305 GSM4689305 GEO Accession GSM4689305 SRX8810063 GSM4689306 SRP273093 PRJNA647773 SRS7077229 GSM4689306 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689306 GSM4689306 GEO Accession GSM4689306 SRX8810064 GSM4689307 SRP273093 PRJNA647773 SRS7077230 GSM4689307 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689307 GSM4689307 GEO Accession GSM4689307 SRX8810065 GSM4689308 SRP273093 PRJNA647773 SRS7077228 GSM4689308 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689308 GSM4689308 GEO Accession GSM4689308 SRX8810066 GSM4689309 SRP273093 PRJNA647773 SRS7077233 GSM4689309 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689309 GSM4689309 GEO Accession GSM4689309 SRX8810067 GSM4689310 SRP273093 PRJNA647773 SRS7077231 GSM4689310 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689310 GSM4689310 GEO Accession GSM4689310 SRX8810068 GSM4689311 SRP273093 PRJNA647773 SRS7077232 GSM4689311 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689311 GSM4689311 GEO Accession GSM4689311 SRX8810069 GSM4689312 SRP273093 PRJNA647773 SRS7077234 GSM4689312 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689312 GSM4689312 GEO Accession GSM4689312 SRX8810070 GSM4689313 SRP273093 PRJNA647773 SRS7077236 GSM4689313 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689313 GSM4689313 GEO Accession GSM4689313 SRX8810071 GSM4689314 SRP273093 PRJNA647773 SRS7077235 GSM4689314 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689314 GSM4689314 GEO Accession GSM4689314 SRX8810072 GSM4689315 SRP273093 PRJNA647773 SRS7077237 GSM4689315 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689315 GSM4689315 GEO Accession GSM4689315 SRX8810073 GSM4689316 SRP273093 PRJNA647773 SRS7077238 GSM4689316 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689316 GSM4689316 GEO Accession GSM4689316 SRX8810074 GSM4689317 SRP273093 PRJNA647773 SRS7077239 GSM4689317 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689317 GSM4689317 GEO Accession GSM4689317 SRX8810075 GSM4689318 SRP273093 PRJNA647773 SRS7077241 GSM4689318 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689318 GSM4689318 GEO Accession GSM4689318 SRX8810076 GSM4689319 SRP273093 PRJNA647773 SRS7077240 GSM4689319 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689319 GSM4689319 GEO Accession GSM4689319 SRX8810077 GSM4689320 SRP273093 PRJNA647773 SRS7077242 GSM4689320 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689320 GSM4689320 GEO Accession GSM4689320 SRX8810078 GSM4689321 SRP273093 PRJNA647773 SRS7077244 GSM4689321 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689321 GSM4689321 GEO Accession GSM4689321 SRX8810079 GSM4689322 SRP273093 PRJNA647773 SRS7077243 GSM4689322 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689322 GSM4689322 GEO Accession GSM4689322 SRX8810080 GSM4689323 SRP273093 PRJNA647773 SRS7077245 GSM4689323 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689323 GSM4689323 GEO Accession GSM4689323 SRX8810081 GSM4689324 SRP273093 PRJNA647773 SRS7077246 GSM4689324 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689324 GSM4689324 GEO Accession GSM4689324 SRX8810082 GSM4689325 SRP273093 PRJNA647773 SRS7077247 GSM4689325 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689325 GSM4689325 GEO Accession GSM4689325 SRX8810083 GSM4689326 SRP273093 PRJNA647773 SRS7077249 GSM4689326 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689326 GSM4689326 GEO Accession GSM4689326 SRX8810084 GSM4689327 SRP273093 PRJNA647773 SRS7077248 GSM4689327 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689327 GSM4689327 GEO Accession GSM4689327 SRX8810085 GSM4689328 SRP273093 PRJNA647773 SRS7077251 GSM4689328 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689328 GSM4689328 GEO Accession GSM4689328 SRX8810086 GSM4689329 SRP273093 PRJNA647773 SRS7077250 GSM4689329 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689329 GSM4689329 GEO Accession GSM4689329 SRX8810087 GSM4689330 SRP273093 PRJNA647773 SRS7077252 GSM4689330 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689330 GSM4689330 GEO Accession GSM4689330 SRX8810088 GSM4689331 SRP273093 PRJNA647773 SRS7077253 GSM4689331 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689331 GSM4689331 GEO Accession GSM4689331 SRX8810089 GSM4689332 SRP273093 PRJNA647773 SRS7077254 GSM4689332 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689332 GSM4689332 GEO Accession GSM4689332 SRX8810090 GSM4689333 SRP273093 PRJNA647773 SRS7077256 GSM4689333 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689333 GSM4689333 GEO Accession GSM4689333 SRX8810091 GSM4689334 SRP273093 PRJNA647773 SRS7077255 GSM4689334 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689334 GSM4689334 GEO Accession GSM4689334 SRX8810092 GSM4689335 SRP273093 PRJNA647773 SRS7077257 GSM4689335 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689335 GSM4689335 GEO Accession GSM4689335 SRX8810093 GSM4689336 SRP273093 PRJNA647773 SRS7077258 GSM4689336 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689336 GSM4689336 GEO Accession GSM4689336 SRX8810094 GSM4689337 SRP273093 PRJNA647773 SRS7077260 GSM4689337 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689337 GSM4689337 GEO Accession GSM4689337 SRX8810095 GSM4689338 SRP273093 PRJNA647773 SRS7077259 GSM4689338 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689338 GSM4689338 GEO Accession GSM4689338 SRX8810096 GSM4689339 SRP273093 PRJNA647773 SRS7077261 GSM4689339 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689339 GSM4689339 GEO Accession GSM4689339 SRX8810097 GSM4689340 SRP273093 PRJNA647773 SRS7077263 GSM4689340 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689340 GSM4689340 GEO Accession GSM4689340 SRX8810098 GSM4689341 SRP273093 PRJNA647773 SRS7077262 GSM4689341 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689341 GSM4689341 GEO Accession GSM4689341 SRX8810099 GSM4689342 SRP273093 PRJNA647773 SRS7077264 GSM4689342 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689342 GSM4689342 GEO Accession GSM4689342 SRX8810100 GSM4689343 SRP273093 PRJNA647773 SRS7077265 GSM4689343 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689343 GSM4689343 GEO Accession GSM4689343 SRX8810101 GSM4689344 SRP273093 PRJNA647773 SRS7077266 GSM4689344 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689344 GSM4689344 GEO Accession GSM4689344 SRX8810102 GSM4689345 SRP273093 PRJNA647773 SRS7077267 GSM4689345 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689345 GSM4689345 GEO Accession GSM4689345 SRX8810103 GSM4689346 SRP273093 PRJNA647773 SRS7077268 GSM4689346 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689346 GSM4689346 GEO Accession GSM4689346 SRX8810104 GSM4689347 SRP273093 PRJNA647773 SRS7077270 GSM4689347 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689347 GSM4689347 GEO Accession GSM4689347 SRX8810105 GSM4689348 SRP273093 PRJNA647773 SRS7077269 GSM4689348 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689348 GSM4689348 GEO Accession GSM4689348 SRX8810106 GSM4689349 SRP273093 PRJNA647773 SRS7077272 GSM4689349 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689349 GSM4689349 GEO Accession GSM4689349 SRX8810107 GSM4689350 SRP273093 PRJNA647773 SRS7077271 GSM4689350 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689350 GSM4689350 GEO Accession GSM4689350 SRX8810108 GSM4689351 SRP273093 PRJNA647773 SRS7077273 GSM4689351 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689351 GSM4689351 GEO Accession GSM4689351 SRX8810109 GSM4689352 SRP273093 PRJNA647773 SRS7077276 GSM4689352 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689352 GSM4689352 GEO Accession GSM4689352 SRX8810110 GSM4689353 SRP273093 PRJNA647773 SRS7077275 GSM4689353 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689353 GSM4689353 GEO Accession GSM4689353 SRX8810111 GSM4689354 SRP273093 PRJNA647773 SRS7077274 GSM4689354 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689354 GSM4689354 GEO Accession GSM4689354 SRX8810112 GSM4689355 SRP273093 PRJNA647773 SRS7077277 GSM4689355 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689355 GSM4689355 GEO Accession GSM4689355 SRX8810113 GSM4689356 SRP273093 PRJNA647773 SRS7077278 GSM4689356 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689356 GSM4689356 GEO Accession GSM4689356 SRX8810114 GSM4689357 SRP273093 PRJNA647773 SRS7077279 GSM4689357 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689357 GSM4689357 GEO Accession GSM4689357 SRX8810115 GSM4689358 SRP273093 PRJNA647773 SRS7077281 GSM4689358 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689358 GSM4689358 GEO Accession GSM4689358 SRX8810116 GSM4689359 SRP273093 PRJNA647773 SRS7077282 GSM4689359 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689359 GSM4689359 GEO Accession GSM4689359 SRX8810117 GSM4689360 SRP273093 PRJNA647773 SRS7077280 GSM4689360 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689360 GSM4689360 GEO Accession GSM4689360 SRX8810118 GSM4689361 SRP273093 PRJNA647773 SRS7077284 GSM4689361 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689361 GSM4689361 GEO Accession GSM4689361 SRX8810119 GSM4689362 SRP273093 PRJNA647773 SRS7077283 GSM4689362 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689362 GSM4689362 GEO Accession GSM4689362 SRX8810120 GSM4689363 SRP273093 PRJNA647773 SRS7077285 GSM4689363 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689363 GSM4689363 GEO Accession GSM4689363 SRX8810121 GSM4689364 SRP273093 PRJNA647773 SRS7077286 GSM4689364 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689364 GSM4689364 GEO Accession GSM4689364 SRX8810122 GSM4689365 SRP273093 PRJNA647773 SRS7077288 GSM4689365 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689365 GSM4689365 GEO Accession GSM4689365 SRX8810123 GSM4689366 SRP273093 PRJNA647773 SRS7077287 GSM4689366 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689366 GSM4689366 GEO Accession GSM4689366 SRX8810124 GSM4689367 SRP273093 PRJNA647773 SRS7077289 GSM4689367 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689367 GSM4689367 GEO Accession GSM4689367 SRX8810125 GSM4689368 SRP273093 PRJNA647773 SRS7077290 GSM4689368 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689368 GSM4689368 GEO Accession GSM4689368 SRX8810126 GSM4689369 SRP273093 PRJNA647773 SRS7077291 GSM4689369 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689369 GSM4689369 GEO Accession GSM4689369 SRX8810127 GSM4689370 SRP273093 PRJNA647773 SRS7077292 GSM4689370 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689370 GSM4689370 GEO Accession GSM4689370 SRX8810128 GSM4689371 SRP273093 PRJNA647773 SRS7077294 GSM4689371 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689371 GSM4689371 GEO Accession GSM4689371 SRX8810129 GSM4689372 SRP273093 PRJNA647773 SRS7077293 GSM4689372 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689372 GSM4689372 GEO Accession GSM4689372 SRX8810130 GSM4689373 SRP273093 PRJNA647773 SRS7077295 GSM4689373 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689373 GSM4689373 GEO Accession GSM4689373 SRX8810131 GSM4689374 SRP273093 PRJNA647773 SRS7077296 GSM4689374 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689374 GSM4689374 GEO Accession GSM4689374 SRX8810132 GSM4689375 SRP273093 PRJNA647773 SRS7076223 GSM4689375 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689375 GSM4689375 GEO Accession GSM4689375 SRX8810133 GSM4689376 SRP273093 PRJNA647773 SRS7077298 GSM4689376 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689376 GSM4689376 GEO Accession GSM4689376 SRX8810134 GSM4689377 SRP273093 PRJNA647773 SRS7077297 GSM4689377 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689377 GSM4689377 GEO Accession GSM4689377 SRX8810135 GSM4689378 SRP273093 PRJNA647773 SRS7077299 GSM4689378 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689378 GSM4689378 GEO Accession GSM4689378 SRX8810136 GSM4689379 SRP273093 PRJNA647773 SRS7077300 GSM4689379 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689379 GSM4689379 GEO Accession GSM4689379 SRX8810137 GSM4689380 SRP273093 PRJNA647773 SRS7077301 GSM4689380 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689380 GSM4689380 GEO Accession GSM4689380 SRX8810138 GSM4689381 SRP273093 PRJNA647773 SRS7077302 GSM4689381 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689381 GSM4689381 GEO Accession GSM4689381 SRX8810139 GSM4689382 SRP273093 PRJNA647773 SRS7077303 GSM4689382 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689382 GSM4689382 GEO Accession GSM4689382 SRX8810140 GSM4689383 SRP273093 PRJNA647773 SRS7077305 GSM4689383 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689383 GSM4689383 GEO Accession GSM4689383 SRX8810141 GSM4689384 SRP273093 PRJNA647773 SRS7077304 GSM4689384 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689384 GSM4689384 GEO Accession GSM4689384 SRX8810142 GSM4689385 SRP273093 PRJNA647773 SRS7077306 GSM4689385 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689385 GSM4689385 GEO Accession GSM4689385 SRX8810143 GSM4689386 SRP273093 PRJNA647773 SRS7077307 GSM4689386 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689386 GSM4689386 GEO Accession GSM4689386 SRX8810144 GSM4689387 SRP273093 PRJNA647773 SRS7077310 GSM4689387 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689387 GSM4689387 GEO Accession GSM4689387 SRX8810145 GSM4689388 SRP273093 PRJNA647773 SRS7077308 GSM4689388 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689388 GSM4689388 GEO Accession GSM4689388 SRX8810146 GSM4689389 SRP273093 PRJNA647773 SRS7077309 GSM4689389 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689389 GSM4689389 GEO Accession GSM4689389 SRX8810147 GSM4689390 SRP273093 PRJNA647773 SRS7077311 GSM4689390 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689390 GSM4689390 GEO Accession GSM4689390 SRX8810148 GSM4689391 SRP273093 PRJNA647773 SRS7077313 GSM4689391 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689391 GSM4689391 GEO Accession GSM4689391 SRX8810149 GSM4689392 SRP273093 PRJNA647773 SRS7077312 GSM4689392 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689392 GSM4689392 GEO Accession GSM4689392 SRX8810150 GSM4689393 SRP273093 PRJNA647773 SRS7077314 GSM4689393 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689393 GSM4689393 GEO Accession GSM4689393 SRX8810151 GSM4689394 SRP273093 PRJNA647773 SRS7077315 GSM4689394 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689394 GSM4689394 GEO Accession GSM4689394 SRX8810152 GSM4689395 SRP273093 PRJNA647773 SRS7077316 GSM4689395 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689395 GSM4689395 GEO Accession GSM4689395 SRX8810153 GSM4689396 SRP273093 PRJNA647773 SRS7077318 GSM4689396 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689396 GSM4689396 GEO Accession GSM4689396 SRX8810154 GSM4689397 SRP273093 PRJNA647773 SRS7077317 GSM4689397 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689397 GSM4689397 GEO Accession GSM4689397 SRX8810155 GSM4689398 SRP273093 PRJNA647773 SRS7077319 GSM4689398 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689398 GSM4689398 GEO Accession GSM4689398 SRX8810156 GSM4689399 SRP273093 PRJNA647773 SRS7077320 GSM4689399 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689399 GSM4689399 GEO Accession GSM4689399 SRX8810157 GSM4689400 SRP273093 PRJNA647773 SRS7077321 GSM4689400 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689400 GSM4689400 GEO Accession GSM4689400 SRX8810158 GSM4689401 SRP273093 PRJNA647773 SRS7077323 GSM4689401 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689401 GSM4689401 GEO Accession GSM4689401 SRX8810159 GSM4689402 SRP273093 PRJNA647773 SRS7077322 GSM4689402 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689402 GSM4689402 GEO Accession GSM4689402 SRX8810160 GSM4689403 SRP273093 PRJNA647773 SRS7077326 GSM4689403 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689403 GSM4689403 GEO Accession GSM4689403 SRX8810161 GSM4689404 SRP273093 PRJNA647773 SRS7077324 GSM4689404 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689404 GSM4689404 GEO Accession GSM4689404 SRX8810162 GSM4689405 SRP273093 PRJNA647773 SRS7077325 GSM4689405 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689405 GSM4689405 GEO Accession GSM4689405 SRX8810163 GSM4689406 SRP273093 PRJNA647773 SRS7077328 GSM4689406 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689406 GSM4689406 GEO Accession GSM4689406 SRX8810164 GSM4689407 SRP273093 PRJNA647773 SRS7077330 GSM4689407 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689407 GSM4689407 GEO Accession GSM4689407 SRX8810165 GSM4689408 SRP273093 PRJNA647773 SRS7077327 GSM4689408 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689408 GSM4689408 GEO Accession GSM4689408 SRX8810166 GSM4689409 SRP273093 PRJNA647773 SRS7077329 GSM4689409 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689409 GSM4689409 GEO Accession GSM4689409 SRX8810167 GSM4689410 SRP273093 PRJNA647773 SRS7077331 GSM4689410 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689410 GSM4689410 GEO Accession GSM4689410 SRX8810168 GSM4689411 SRP273093 PRJNA647773 SRS7077332 GSM4689411 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689411 GSM4689411 GEO Accession GSM4689411 SRX8810169 GSM4689412 SRP273093 PRJNA647773 SRS7077333 GSM4689412 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689412 GSM4689412 GEO Accession GSM4689412 SRX8810170 GSM4689413 SRP273093 PRJNA647773 SRS7077334 GSM4689413 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689413 GSM4689413 GEO Accession GSM4689413 SRX8810171 GSM4689414 SRP273093 PRJNA647773 SRS7077335 GSM4689414 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689414 GSM4689414 GEO Accession GSM4689414 SRX8810172 GSM4689415 SRP273093 PRJNA647773 SRS7077338 GSM4689415 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689415 GSM4689415 GEO Accession GSM4689415 SRX8810173 GSM4689416 SRP273093 PRJNA647773 SRS7077336 GSM4689416 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689416 GSM4689416 GEO Accession GSM4689416 SRX8810174 GSM4689417 SRP273093 PRJNA647773 SRS7077337 GSM4689417 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689417 GSM4689417 GEO Accession GSM4689417 SRX8810175 GSM4689418 SRP273093 PRJNA647773 SRS7077339 GSM4689418 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689418 GSM4689418 GEO Accession GSM4689418 SRX8810176 GSM4689419 SRP273093 PRJNA647773 SRS7077340 GSM4689419 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689419 GSM4689419 GEO Accession GSM4689419 SRX8810177 GSM4689420 SRP273093 PRJNA647773 SRS7077341 GSM4689420 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689420 GSM4689420 GEO Accession GSM4689420 SRX8810178 GSM4689421 SRP273093 PRJNA647773 SRS7077342 GSM4689421 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689421 GSM4689421 GEO Accession GSM4689421 SRX8810179 GSM4689422 SRP273093 PRJNA647773 SRS7077343 GSM4689422 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689422 GSM4689422 GEO Accession GSM4689422 SRX8810180 GSM4689423 SRP273093 PRJNA647773 SRS7077344 GSM4689423 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689423 GSM4689423 GEO Accession GSM4689423 SRX8810181 GSM4689424 SRP273093 PRJNA647773 SRS7077346 GSM4689424 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689424 GSM4689424 GEO Accession GSM4689424 SRX8810182 GSM4689425 SRP273093 PRJNA647773 SRS7077345 GSM4689425 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689425 GSM4689425 GEO Accession GSM4689425 SRX8810183 GSM4689426 SRP273093 PRJNA647773 SRS7077347 GSM4689426 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689426 GSM4689426 GEO Accession GSM4689426 SRX8810184 GSM4689427 SRP273093 PRJNA647773 SRS7077349 GSM4689427 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689427 GSM4689427 GEO Accession GSM4689427 SRX8810185 GSM4689428 SRP273093 PRJNA647773 SRS7077351 GSM4689428 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689428 GSM4689428 GEO Accession GSM4689428 SRX8810186 GSM4689429 SRP273093 PRJNA647773 SRS7077348 GSM4689429 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689429 GSM4689429 GEO Accession GSM4689429 SRX8810187 GSM4689430 SRP273093 PRJNA647773 SRS7077350 GSM4689430 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689430 GSM4689430 GEO Accession GSM4689430 SRX8810188 GSM4689431 SRP273093 PRJNA647773 SRS7077353 GSM4689431 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689431 GSM4689431 GEO Accession GSM4689431 SRX8810189 GSM4689432 SRP273093 PRJNA647773 SRS7077352 GSM4689432 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689432 GSM4689432 GEO Accession GSM4689432 SRX8810190 GSM4689433 SRP273093 PRJNA647773 SRS7077354 GSM4689433 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689433 GSM4689433 GEO Accession GSM4689433 SRX8810191 GSM4689434 SRP273093 PRJNA647773 SRS7077356 GSM4689434 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689434 GSM4689434 GEO Accession GSM4689434 SRX8810192 GSM4689435 SRP273093 PRJNA647773 SRS7077355 GSM4689435 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689435 GSM4689435 GEO Accession GSM4689435 SRX8810875 GSM4686796 SRP273093 PRJNA647773 SRS7074921 GSM4686796 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686796 GSM4686796 GEO Accession GSM4686796 SRX8810876 GSM4686797 SRP273093 PRJNA647773 SRS7074920 GSM4686797 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686797 GSM4686797 GEO Accession GSM4686797 SRX8810877 GSM4686798 SRP273093 PRJNA647773 SRS7074922 GSM4686798 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686798 GSM4686798 GEO Accession GSM4686798 SRX8810878 GSM4686799 SRP273093 PRJNA647773 SRS7074923 GSM4686799 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686799 GSM4686799 GEO Accession GSM4686799 SRX8810879 GSM4686800 SRP273093 PRJNA647773 SRS7074924 GSM4686800 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686800 GSM4686800 GEO Accession GSM4686800 SRX8810880 GSM4686801 SRP273093 PRJNA647773 SRS7074925 GSM4686801 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686801 GSM4686801 GEO Accession GSM4686801 SRX8810881 GSM4686802 SRP273093 PRJNA647773 SRS7074926 GSM4686802 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686802 GSM4686802 GEO Accession GSM4686802 SRX8810882 GSM4686803 SRP273093 PRJNA647773 SRS7074927 GSM4686803 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686803 GSM4686803 GEO Accession GSM4686803 SRX8810883 GSM4686804 SRP273093 PRJNA647773 SRS7074928 GSM4686804 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686804 GSM4686804 GEO Accession GSM4686804 SRX8810884 GSM4686805 SRP273093 PRJNA647773 SRS7074929 GSM4686805 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686805 GSM4686805 GEO Accession GSM4686805 SRX8810885 GSM4686806 SRP273093 PRJNA647773 SRS7074931 GSM4686806 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686806 GSM4686806 GEO Accession GSM4686806 SRX8810886 GSM4686807 SRP273093 PRJNA647773 SRS7074930 GSM4686807 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686807 GSM4686807 GEO Accession GSM4686807 SRX8810887 GSM4686808 SRP273093 PRJNA647773 SRS7074932 GSM4686808 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686808 GSM4686808 GEO Accession GSM4686808 SRX8810888 GSM4686809 SRP273093 PRJNA647773 SRS7074934 GSM4686809 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686809 GSM4686809 GEO Accession GSM4686809 SRX8810889 GSM4686810 SRP273093 PRJNA647773 SRS7074933 GSM4686810 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686810 GSM4686810 GEO Accession GSM4686810 SRX8810890 GSM4686811 SRP273093 PRJNA647773 SRS7074935 GSM4686811 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686811 GSM4686811 GEO Accession GSM4686811 SRX8810891 GSM4686812 SRP273093 PRJNA647773 SRS7074936 GSM4686812 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686812 GSM4686812 GEO Accession GSM4686812 SRX8810892 GSM4686813 SRP273093 PRJNA647773 SRS7074937 GSM4686813 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686813 GSM4686813 GEO Accession GSM4686813 SRX8810893 GSM4686814 SRP273093 PRJNA647773 SRS7074938 GSM4686814 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686814 GSM4686814 GEO Accession GSM4686814 SRX8810894 GSM4686815 SRP273093 PRJNA647773 SRS7074939 GSM4686815 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686815 GSM4686815 GEO Accession GSM4686815 SRX8810895 GSM4686816 SRP273093 PRJNA647773 SRS7074940 GSM4686816 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686816 GSM4686816 GEO Accession GSM4686816 SRX8810896 GSM4686817 SRP273093 PRJNA647773 SRS7074941 GSM4686817 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686817 GSM4686817 GEO Accession GSM4686817 SRX8810897 GSM4686818 SRP273093 PRJNA647773 SRS7074942 GSM4686818 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686818 GSM4686818 GEO Accession GSM4686818 SRX8810898 GSM4686819 SRP273093 PRJNA647773 SRS7074943 GSM4686819 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686819 GSM4686819 GEO Accession GSM4686819 SRX8810899 GSM4686820 SRP273093 PRJNA647773 SRS7074944 GSM4686820 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686820 GSM4686820 GEO Accession GSM4686820 SRX8810900 GSM4686821 SRP273093 PRJNA647773 SRS7074945 GSM4686821 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686821 GSM4686821 GEO Accession GSM4686821 SRX8810901 GSM4686822 SRP273093 PRJNA647773 SRS7074946 GSM4686822 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686822 GSM4686822 GEO Accession GSM4686822 SRX8810902 GSM4686823 SRP273093 PRJNA647773 SRS7074947 GSM4686823 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686823 GSM4686823 GEO Accession GSM4686823 SRX8810903 GSM4686824 SRP273093 PRJNA647773 SRS7074949 GSM4686824 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686824 GSM4686824 GEO Accession GSM4686824 SRX8810904 GSM4686825 SRP273093 PRJNA647773 SRS7074948 GSM4686825 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686825 GSM4686825 GEO Accession GSM4686825 SRX8810905 GSM4686826 SRP273093 PRJNA647773 SRS7074950 GSM4686826 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686826 GSM4686826 GEO Accession GSM4686826 SRX8810906 GSM4686827 SRP273093 PRJNA647773 SRS7074951 GSM4686827 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686827 GSM4686827 GEO Accession GSM4686827 SRX8810907 GSM4686828 SRP273093 PRJNA647773 SRS7074952 GSM4686828 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686828 GSM4686828 GEO Accession GSM4686828 SRX8810908 GSM4686829 SRP273093 PRJNA647773 SRS7074953 GSM4686829 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686829 GSM4686829 GEO Accession GSM4686829 SRX8810909 GSM4686830 SRP273093 PRJNA647773 SRS7074954 GSM4686830 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686830 GSM4686830 GEO Accession GSM4686830 SRX8810910 GSM4686831 SRP273093 PRJNA647773 SRS7074955 GSM4686831 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686831 GSM4686831 GEO Accession GSM4686831 SRX8810911 GSM4686832 SRP273093 PRJNA647773 SRS7074956 GSM4686832 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686832 GSM4686832 GEO Accession GSM4686832 SRX8810912 GSM4686833 SRP273093 PRJNA647773 SRS7074957 GSM4686833 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686833 GSM4686833 GEO Accession GSM4686833 SRX8810913 GSM4686834 SRP273093 PRJNA647773 SRS7074958 GSM4686834 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686834 GSM4686834 GEO Accession GSM4686834 SRX8810914 GSM4686835 SRP273093 PRJNA647773 SRS7074959 GSM4686835 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686835 GSM4686835 GEO Accession GSM4686835 SRX8810915 GSM4686836 SRP273093 PRJNA647773 SRS7074960 GSM4686836 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686836 GSM4686836 GEO Accession GSM4686836 SRX8810916 GSM4686837 SRP273093 PRJNA647773 SRS7074961 GSM4686837 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686837 GSM4686837 GEO Accession GSM4686837 SRX8810917 GSM4686838 SRP273093 PRJNA647773 SRS7074962 GSM4686838 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686838 GSM4686838 GEO Accession GSM4686838 SRX8810918 GSM4686839 SRP273093 PRJNA647773 SRS7074963 GSM4686839 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686839 GSM4686839 GEO Accession GSM4686839 SRX8810919 GSM4686840 SRP273093 PRJNA647773 SRS7074964 GSM4686840 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686840 GSM4686840 GEO Accession GSM4686840 SRX8810920 GSM4686841 SRP273093 PRJNA647773 SRS7074966 GSM4686841 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686841 GSM4686841 GEO Accession GSM4686841 SRX8810921 GSM4686842 SRP273093 PRJNA647773 SRS7074965 GSM4686842 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686842 GSM4686842 GEO Accession GSM4686842 SRX8810922 GSM4686843 SRP273093 PRJNA647773 SRS7074967 GSM4686843 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686843 GSM4686843 GEO Accession GSM4686843 SRX8810923 GSM4686844 SRP273093 PRJNA647773 SRS7074968 GSM4686844 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686844 GSM4686844 GEO Accession GSM4686844 SRX8810924 GSM4686845 SRP273093 PRJNA647773 SRS7074969 GSM4686845 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686845 GSM4686845 GEO Accession GSM4686845 SRX8810925 GSM4686846 SRP273093 PRJNA647773 SRS7074970 GSM4686846 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686846 GSM4686846 GEO Accession GSM4686846 SRX8810926 GSM4686847 SRP273093 PRJNA647773 SRS7074971 GSM4686847 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686847 GSM4686847 GEO Accession GSM4686847 SRX8810927 GSM4686848 SRP273093 PRJNA647773 SRS7074972 GSM4686848 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686848 GSM4686848 GEO Accession GSM4686848 SRX8810928 GSM4686849 SRP273093 PRJNA647773 SRS7074973 GSM4686849 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686849 GSM4686849 GEO Accession GSM4686849 SRX8810929 GSM4686850 SRP273093 PRJNA647773 SRS7074974 GSM4686850 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686850 GSM4686850 GEO Accession GSM4686850 SRX8810930 GSM4686851 SRP273093 PRJNA647773 SRS7074975 GSM4686851 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686851 GSM4686851 GEO Accession GSM4686851 SRX8810931 GSM4686852 SRP273093 PRJNA647773 SRS7074977 GSM4686852 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686852 GSM4686852 GEO Accession GSM4686852 SRX8810932 GSM4686853 SRP273093 PRJNA647773 SRS7074976 GSM4686853 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686853 GSM4686853 GEO Accession GSM4686853 SRX8810933 GSM4686854 SRP273093 PRJNA647773 SRS7074978 GSM4686854 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686854 GSM4686854 GEO Accession GSM4686854 SRX8810934 GSM4686855 SRP273093 PRJNA647773 SRS7074979 GSM4686855 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686855 GSM4686855 GEO Accession GSM4686855 SRX8810935 GSM4686856 SRP273093 PRJNA647773 SRS7074980 GSM4686856 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686856 GSM4686856 GEO Accession GSM4686856 SRX8810936 GSM4686857 SRP273093 PRJNA647773 SRS7074981 GSM4686857 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686857 GSM4686857 GEO Accession GSM4686857 SRX8810937 GSM4686858 SRP273093 PRJNA647773 SRS7074982 GSM4686858 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686858 GSM4686858 GEO Accession GSM4686858 SRX8810938 GSM4686859 SRP273093 PRJNA647773 SRS7074983 GSM4686859 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686859 GSM4686859 GEO Accession GSM4686859 SRX8810939 GSM4686860 SRP273093 PRJNA647773 SRS7074984 GSM4686860 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686860 GSM4686860 GEO Accession GSM4686860 SRX8810940 GSM4686861 SRP273093 PRJNA647773 SRS7074985 GSM4686861 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686861 GSM4686861 GEO Accession GSM4686861 SRX8810941 GSM4686862 SRP273093 PRJNA647773 SRS7074986 GSM4686862 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686862 GSM4686862 GEO Accession GSM4686862 SRX8810942 GSM4686863 SRP273093 PRJNA647773 SRS7074987 GSM4686863 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686863 GSM4686863 GEO Accession GSM4686863 SRX8810943 GSM4686864 SRP273093 PRJNA647773 SRS7074988 GSM4686864 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686864 GSM4686864 GEO Accession GSM4686864 SRX8810944 GSM4686865 SRP273093 PRJNA647773 SRS7074989 GSM4686865 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686865 GSM4686865 GEO Accession GSM4686865 SRX8810945 GSM4686866 SRP273093 PRJNA647773 SRS7074990 GSM4686866 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686866 GSM4686866 GEO Accession GSM4686866 SRX8810946 GSM4686867 SRP273093 PRJNA647773 SRS7074991 GSM4686867 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686867 GSM4686867 GEO Accession GSM4686867 SRX8810947 GSM4686868 SRP273093 PRJNA647773 SRS7074992 GSM4686868 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686868 GSM4686868 GEO Accession GSM4686868 SRX8810948 GSM4686869 SRP273093 PRJNA647773 SRS7074993 GSM4686869 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686869 GSM4686869 GEO Accession GSM4686869 SRX8810949 GSM4686870 SRP273093 PRJNA647773 SRS7074994 GSM4686870 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686870 GSM4686870 GEO Accession GSM4686870 SRX8810950 GSM4686871 SRP273093 PRJNA647773 SRS7074995 GSM4686871 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686871 GSM4686871 GEO Accession GSM4686871 SRX8810951 GSM4686872 SRP273093 PRJNA647773 SRS7074996 GSM4686872 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686872 GSM4686872 GEO Accession GSM4686872 SRX8810952 GSM4686873 SRP273093 PRJNA647773 SRS7074997 GSM4686873 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686873 GSM4686873 GEO Accession GSM4686873 SRX8810953 GSM4686874 SRP273093 PRJNA647773 SRS7074999 GSM4686874 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686874 GSM4686874 GEO Accession GSM4686874 SRX8810954 GSM4686875 SRP273093 PRJNA647773 SRS7074998 GSM4686875 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686875 GSM4686875 GEO Accession GSM4686875 SRX8810955 GSM4686876 SRP273093 PRJNA647773 SRS7075000 GSM4686876 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686876 GSM4686876 GEO Accession GSM4686876 SRX8810956 GSM4686877 SRP273093 PRJNA647773 SRS7075001 GSM4686877 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686877 GSM4686877 GEO Accession GSM4686877 SRX8810957 GSM4686878 SRP273093 PRJNA647773 SRS7075002 GSM4686878 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686878 GSM4686878 GEO Accession GSM4686878 SRX8810958 GSM4686879 SRP273093 PRJNA647773 SRS7075003 GSM4686879 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686879 GSM4686879 GEO Accession GSM4686879 SRX8810959 GSM4686880 SRP273093 PRJNA647773 SRS7075004 GSM4686880 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686880 GSM4686880 GEO Accession GSM4686880 SRX8810960 GSM4686881 SRP273093 PRJNA647773 SRS7075005 GSM4686881 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686881 GSM4686881 GEO Accession GSM4686881 SRX8810961 GSM4686882 SRP273093 PRJNA647773 SRS7075006 GSM4686882 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686882 GSM4686882 GEO Accession GSM4686882 SRX8810962 GSM4686883 SRP273093 PRJNA647773 SRS7075007 GSM4686883 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686883 GSM4686883 GEO Accession GSM4686883 SRX8810963 GSM4686884 SRP273093 PRJNA647773 SRS7075008 GSM4686884 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686884 GSM4686884 GEO Accession GSM4686884 SRX8810964 GSM4686885 SRP273093 PRJNA647773 SRS7075009 GSM4686885 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686885 GSM4686885 GEO Accession GSM4686885 SRX8810965 GSM4686886 SRP273093 PRJNA647773 SRS7075010 GSM4686886 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686886 GSM4686886 GEO Accession GSM4686886 SRX8810966 GSM4686887 SRP273093 PRJNA647773 SRS7075011 GSM4686887 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686887 GSM4686887 GEO Accession GSM4686887 SRX8810967 GSM4686888 SRP273093 PRJNA647773 SRS7075013 GSM4686888 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686888 GSM4686888 GEO Accession GSM4686888 SRX8810968 GSM4686889 SRP273093 PRJNA647773 SRS7075012 GSM4686889 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686889 GSM4686889 GEO Accession GSM4686889 SRX8810969 GSM4686890 SRP273093 PRJNA647773 SRS7075014 GSM4686890 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686890 GSM4686890 GEO Accession GSM4686890 SRX8810970 GSM4686891 SRP273093 PRJNA647773 SRS7075015 GSM4686891 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686891 GSM4686891 GEO Accession GSM4686891 SRX8810971 GSM4686892 SRP273093 PRJNA647773 SRS7075016 GSM4686892 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686892 GSM4686892 GEO Accession GSM4686892 SRX8810972 GSM4686893 SRP273093 PRJNA647773 SRS7075017 GSM4686893 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686893 GSM4686893 GEO Accession GSM4686893 SRX8810973 GSM4686894 SRP273093 PRJNA647773 SRS7075018 GSM4686894 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686894 GSM4686894 GEO Accession GSM4686894 SRX8810974 GSM4686895 SRP273093 PRJNA647773 SRS7075019 GSM4686895 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686895 GSM4686895 GEO Accession GSM4686895 SRX8810975 GSM4686896 SRP273093 PRJNA647773 SRS7075020 GSM4686896 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686896 GSM4686896 GEO Accession GSM4686896 SRX8810976 GSM4686897 SRP273093 PRJNA647773 SRS7075021 GSM4686897 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686897 GSM4686897 GEO Accession GSM4686897 SRX8810977 GSM4686898 SRP273093 PRJNA647773 SRS7075022 GSM4686898 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686898 GSM4686898 GEO Accession GSM4686898 SRX8810978 GSM4686899 SRP273093 PRJNA647773 SRS7075023 GSM4686899 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686899 GSM4686899 GEO Accession GSM4686899 SRX8810979 GSM4686900 SRP273093 PRJNA647773 SRS7075024 GSM4686900 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686900 GSM4686900 GEO Accession GSM4686900 SRX8810980 GSM4686901 SRP273093 PRJNA647773 SRS7075025 GSM4686901 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686901 GSM4686901 GEO Accession GSM4686901 SRX8810981 GSM4686902 SRP273093 PRJNA647773 SRS7075026 GSM4686902 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686902 GSM4686902 GEO Accession GSM4686902 SRX8810982 GSM4686903 SRP273093 PRJNA647773 SRS7075027 GSM4686903 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686903 GSM4686903 GEO Accession GSM4686903 SRX8810983 GSM4686904 SRP273093 PRJNA647773 SRS7075029 GSM4686904 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686904 GSM4686904 GEO Accession GSM4686904 SRX8810984 GSM4686905 SRP273093 PRJNA647773 SRS7075028 GSM4686905 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686905 GSM4686905 GEO Accession GSM4686905 SRX8810985 GSM4686906 SRP273093 PRJNA647773 SRS7075030 GSM4686906 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686906 GSM4686906 GEO Accession GSM4686906 SRX8810986 GSM4686907 SRP273093 PRJNA647773 SRS7075031 GSM4686907 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686907 GSM4686907 GEO Accession GSM4686907 SRX8810987 GSM4686908 SRP273093 PRJNA647773 SRS7075032 GSM4686908 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686908 GSM4686908 GEO Accession GSM4686908 SRX8810988 GSM4686909 SRP273093 PRJNA647773 SRS7075033 GSM4686909 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686909 GSM4686909 GEO Accession GSM4686909 SRX8810989 GSM4686910 SRP273093 PRJNA647773 SRS7075034 GSM4686910 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686910 GSM4686910 GEO Accession GSM4686910 SRX8810990 GSM4686911 SRP273093 PRJNA647773 SRS7075037 GSM4686911 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686911 GSM4686911 GEO Accession GSM4686911 SRX8810991 GSM4686912 SRP273093 PRJNA647773 SRS7075036 GSM4686912 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686912 GSM4686912 GEO Accession GSM4686912 SRX8810992 GSM4686913 SRP273093 PRJNA647773 SRS7075035 GSM4686913 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686913 GSM4686913 GEO Accession GSM4686913 SRX8810993 GSM4686914 SRP273093 PRJNA647773 SRS7075038 GSM4686914 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686914 GSM4686914 GEO Accession GSM4686914 SRX8810994 GSM4686915 SRP273093 PRJNA647773 SRS7075039 GSM4686915 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686915 GSM4686915 GEO Accession GSM4686915 SRX8810995 GSM4686916 SRP273093 PRJNA647773 SRS7075040 GSM4686916 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686916 GSM4686916 GEO Accession GSM4686916 SRX8810996 GSM4686917 SRP273093 PRJNA647773 SRS7075041 GSM4686917 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686917 GSM4686917 GEO Accession GSM4686917 SRX8810997 GSM4686918 SRP273093 PRJNA647773 SRS7075042 GSM4686918 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686918 GSM4686918 GEO Accession GSM4686918 SRX8810998 GSM4686919 SRP273093 PRJNA647773 SRS7075043 GSM4686919 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686919 GSM4686919 GEO Accession GSM4686919 SRX8810999 GSM4686920 SRP273093 PRJNA647773 SRS7075044 GSM4686920 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686920 GSM4686920 GEO Accession GSM4686920 SRX8811000 GSM4686921 SRP273093 PRJNA647773 SRS7075045 GSM4686921 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686921 GSM4686921 GEO Accession GSM4686921 SRX8811001 GSM4686922 SRP273093 PRJNA647773 SRS7075046 GSM4686922 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686922 GSM4686922 GEO Accession GSM4686922 SRX8811002 GSM4686923 SRP273093 PRJNA647773 SRS7075047 GSM4686923 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686923 GSM4686923 GEO Accession GSM4686923 SRX8811003 GSM4686924 SRP273093 PRJNA647773 SRS7075048 GSM4686924 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686924 GSM4686924 GEO Accession GSM4686924 SRX8811004 GSM4686925 SRP273093 PRJNA647773 SRS7075049 GSM4686925 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686925 GSM4686925 GEO Accession GSM4686925 SRX8811005 GSM4686926 SRP273093 PRJNA647773 SRS7075050 GSM4686926 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686926 GSM4686926 GEO Accession GSM4686926 SRX8811006 GSM4686927 SRP273093 PRJNA647773 SRS7075051 GSM4686927 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686927 GSM4686927 GEO Accession GSM4686927 SRX8811007 GSM4686928 SRP273093 PRJNA647773 SRS7075052 GSM4686928 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686928 GSM4686928 GEO Accession GSM4686928 SRX8811008 GSM4686929 SRP273093 PRJNA647773 SRS7075053 GSM4686929 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686929 GSM4686929 GEO Accession GSM4686929 SRX8811009 GSM4686930 SRP273093 PRJNA647773 SRS7075054 GSM4686930 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686930 GSM4686930 GEO Accession GSM4686930 SRX8811010 GSM4686931 SRP273093 PRJNA647773 SRS7075055 GSM4686931 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686931 GSM4686931 GEO Accession GSM4686931 SRX8811011 GSM4686932 SRP273093 PRJNA647773 SRS7075056 GSM4686932 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686932 GSM4686932 GEO Accession GSM4686932 SRX8811012 GSM4686933 SRP273093 PRJNA647773 SRS7075057 GSM4686933 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686933 GSM4686933 GEO Accession GSM4686933 SRX8811013 GSM4686934 SRP273093 PRJNA647773 SRS7075058 GSM4686934 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686934 GSM4686934 GEO Accession GSM4686934 SRX8811014 GSM4686935 SRP273093 PRJNA647773 SRS7075059 GSM4686935 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686935 GSM4686935 GEO Accession GSM4686935 SRX8811015 GSM4686936 SRP273093 PRJNA647773 SRS7075060 GSM4686936 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686936 GSM4686936 GEO Accession GSM4686936 SRX8811016 GSM4686937 SRP273093 PRJNA647773 SRS7075061 GSM4686937 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686937 GSM4686937 GEO Accession GSM4686937 SRX8811017 GSM4686938 SRP273093 PRJNA647773 SRS7075062 GSM4686938 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686938 GSM4686938 GEO Accession GSM4686938 SRX8811018 GSM4686939 SRP273093 PRJNA647773 SRS7075063 GSM4686939 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686939 GSM4686939 GEO Accession GSM4686939 SRX8811019 GSM4686940 SRP273093 PRJNA647773 SRS7075064 GSM4686940 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686940 GSM4686940 GEO Accession GSM4686940 SRX8811020 GSM4686941 SRP273093 PRJNA647773 SRS7075065 GSM4686941 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686941 GSM4686941 GEO Accession GSM4686941 SRX8811021 GSM4686942 SRP273093 PRJNA647773 SRS7075066 GSM4686942 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686942 GSM4686942 GEO Accession GSM4686942 SRX8811022 GSM4686943 SRP273093 PRJNA647773 SRS7075067 GSM4686943 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686943 GSM4686943 GEO Accession GSM4686943 SRX8811023 GSM4686944 SRP273093 PRJNA647773 SRS7075068 GSM4686944 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686944 GSM4686944 GEO Accession GSM4686944 SRX8811024 GSM4686945 SRP273093 PRJNA647773 SRS7075069 GSM4686945 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686945 GSM4686945 GEO Accession GSM4686945 SRX8811025 GSM4686946 SRP273093 PRJNA647773 SRS7075070 GSM4686946 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686946 GSM4686946 GEO Accession GSM4686946 SRX8811026 GSM4686947 SRP273093 PRJNA647773 SRS7075071 GSM4686947 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686947 GSM4686947 GEO Accession GSM4686947 SRX8811027 GSM4686948 SRP273093 PRJNA647773 SRS7075072 GSM4686948 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686948 GSM4686948 GEO Accession GSM4686948 SRX8811028 GSM4686949 SRP273093 PRJNA647773 SRS7075073 GSM4686949 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686949 GSM4686949 GEO Accession GSM4686949 SRX8811029 GSM4686950 SRP273093 PRJNA647773 SRS7075074 GSM4686950 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686950 GSM4686950 GEO Accession GSM4686950 SRX8811030 GSM4686951 SRP273093 PRJNA647773 SRS7075075 GSM4686951 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686951 GSM4686951 GEO Accession GSM4686951 SRX8811031 GSM4686952 SRP273093 PRJNA647773 SRS7075076 GSM4686952 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686952 GSM4686952 GEO Accession GSM4686952 SRX8811032 GSM4686953 SRP273093 PRJNA647773 SRS7075078 GSM4686953 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686953 GSM4686953 GEO Accession GSM4686953 SRX8811033 GSM4686954 SRP273093 PRJNA647773 SRS7075077 GSM4686954 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686954 GSM4686954 GEO Accession GSM4686954 SRX8811034 GSM4686955 SRP273093 PRJNA647773 SRS7075080 GSM4686955 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686955 GSM4686955 GEO Accession GSM4686955 SRX8811035 GSM4686956 SRP273093 PRJNA647773 SRS7075079 GSM4686956 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686956 GSM4686956 GEO Accession GSM4686956 SRX8811036 GSM4686957 SRP273093 PRJNA647773 SRS7075081 GSM4686957 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686957 GSM4686957 GEO Accession GSM4686957 SRX8811037 GSM4686958 SRP273093 PRJNA647773 SRS7075082 GSM4686958 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686958 GSM4686958 GEO Accession GSM4686958 SRX8811038 GSM4686959 SRP273093 PRJNA647773 SRS7075084 GSM4686959 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686959 GSM4686959 GEO Accession GSM4686959 SRX8811039 GSM4686960 SRP273093 PRJNA647773 SRS7075083 GSM4686960 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686960 GSM4686960 GEO Accession GSM4686960 SRX8811040 GSM4686961 SRP273093 PRJNA647773 SRS7075085 GSM4686961 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686961 GSM4686961 GEO Accession GSM4686961 SRX8811041 GSM4686962 SRP273093 PRJNA647773 SRS7075086 GSM4686962 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686962 GSM4686962 GEO Accession GSM4686962 SRX8811042 GSM4686963 SRP273093 PRJNA647773 SRS7075087 GSM4686963 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686963 GSM4686963 GEO Accession GSM4686963 SRX8811043 GSM4686964 SRP273093 PRJNA647773 SRS7075088 GSM4686964 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686964 GSM4686964 GEO Accession GSM4686964 SRX8811044 GSM4686965 SRP273093 PRJNA647773 SRS7075089 GSM4686965 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686965 GSM4686965 GEO Accession GSM4686965 SRX8811045 GSM4686966 SRP273093 PRJNA647773 SRS7075090 GSM4686966 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686966 GSM4686966 GEO Accession GSM4686966 SRX8811046 GSM4686967 SRP273093 PRJNA647773 SRS7075091 GSM4686967 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686967 GSM4686967 GEO Accession GSM4686967 SRX8811047 GSM4686968 SRP273093 PRJNA647773 SRS7075092 GSM4686968 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686968 GSM4686968 GEO Accession GSM4686968 SRX8811048 GSM4686969 SRP273093 PRJNA647773 SRS7075093 GSM4686969 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686969 GSM4686969 GEO Accession GSM4686969 SRX8811049 GSM4686970 SRP273093 PRJNA647773 SRS7075094 GSM4686970 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686970 GSM4686970 GEO Accession GSM4686970 SRX8811050 GSM4686971 SRP273093 PRJNA647773 SRS7075095 GSM4686971 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686971 GSM4686971 GEO Accession GSM4686971 SRX8811051 GSM4686972 SRP273093 PRJNA647773 SRS7075097 GSM4686972 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686972 GSM4686972 GEO Accession GSM4686972 SRX8811052 GSM4686973 SRP273093 PRJNA647773 SRS7075096 GSM4686973 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686973 GSM4686973 GEO Accession GSM4686973 SRX8811053 GSM4686974 SRP273093 PRJNA647773 SRS7075098 GSM4686974 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686974 GSM4686974 GEO Accession GSM4686974 SRX8811054 GSM4686975 SRP273093 PRJNA647773 SRS7075099 GSM4686975 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686975 GSM4686975 GEO Accession GSM4686975 SRX8811055 GSM4686976 SRP273093 PRJNA647773 SRS7075100 GSM4686976 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686976 GSM4686976 GEO Accession GSM4686976 SRX8811056 GSM4686977 SRP273093 PRJNA647773 SRS7075101 GSM4686977 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686977 GSM4686977 GEO Accession GSM4686977 SRX8811057 GSM4686978 SRP273093 PRJNA647773 SRS7075102 GSM4686978 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686978 GSM4686978 GEO Accession GSM4686978 SRX8811058 GSM4686979 SRP273093 PRJNA647773 SRS7075103 GSM4686979 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686979 GSM4686979 GEO Accession GSM4686979 SRX8811059 GSM4686980 SRP273093 PRJNA647773 SRS7075104 GSM4686980 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686980 GSM4686980 GEO Accession GSM4686980 SRX8811060 GSM4686981 SRP273093 PRJNA647773 SRS7075105 GSM4686981 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686981 GSM4686981 GEO Accession GSM4686981 SRX8811061 GSM4686982 SRP273093 PRJNA647773 SRS7075106 GSM4686982 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686982 GSM4686982 GEO Accession GSM4686982 SRX8811062 GSM4686983 SRP273093 PRJNA647773 SRS7075107 GSM4686983 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686983 GSM4686983 GEO Accession GSM4686983 SRX8811063 GSM4686984 SRP273093 PRJNA647773 SRS7075108 GSM4686984 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686984 GSM4686984 GEO Accession GSM4686984 SRX8811064 GSM4686985 SRP273093 PRJNA647773 SRS7075109 GSM4686985 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686985 GSM4686985 GEO Accession GSM4686985 SRX8811065 GSM4686986 SRP273093 PRJNA647773 SRS7075110 GSM4686986 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686986 GSM4686986 GEO Accession GSM4686986 SRX8811066 GSM4686987 SRP273093 PRJNA647773 SRS7075111 GSM4686987 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686987 GSM4686987 GEO Accession GSM4686987 SRX8811067 GSM4686988 SRP273093 PRJNA647773 SRS7075112 GSM4686988 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686988 GSM4686988 GEO Accession GSM4686988 SRX8811068 GSM4686989 SRP273093 PRJNA647773 SRS7075113 GSM4686989 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686989 GSM4686989 GEO Accession GSM4686989 SRX8811069 GSM4686990 SRP273093 PRJNA647773 SRS7075114 GSM4686990 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686990 GSM4686990 GEO Accession GSM4686990 SRX8811070 GSM4686991 SRP273093 PRJNA647773 SRS7075115 GSM4686991 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686991 GSM4686991 GEO Accession GSM4686991 SRX8811071 GSM4686992 SRP273093 PRJNA647773 SRS7075116 GSM4686992 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686992 GSM4686992 GEO Accession GSM4686992 SRX8811072 GSM4686993 SRP273093 PRJNA647773 SRS7075117 GSM4686993 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686993 GSM4686993 GEO Accession GSM4686993 SRX8811073 GSM4686994 SRP273093 PRJNA647773 SRS7075118 GSM4686994 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686994 GSM4686994 GEO Accession GSM4686994 SRX8811074 GSM4686995 SRP273093 PRJNA647773 SRS7075120 GSM4686995 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686995 GSM4686995 GEO Accession GSM4686995 SRX8811075 GSM4686996 SRP273093 PRJNA647773 SRS7075119 GSM4686996 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686996 GSM4686996 GEO Accession GSM4686996 SRX8811076 GSM4686997 SRP273093 PRJNA647773 SRS7075121 GSM4686997 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686997 GSM4686997 GEO Accession GSM4686997 SRX8811077 GSM4686998 SRP273093 PRJNA647773 SRS7075122 GSM4686998 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686998 GSM4686998 GEO Accession GSM4686998 SRX8811078 GSM4686999 SRP273093 PRJNA647773 SRS7075123 GSM4686999 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304686999 GSM4686999 GEO Accession GSM4686999 SRX8811079 GSM4687000 SRP273093 PRJNA647773 SRS7075124 GSM4687000 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687000 GSM4687000 GEO Accession GSM4687000 SRX8811080 GSM4687001 SRP273093 PRJNA647773 SRS7075125 GSM4687001 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687001 GSM4687001 GEO Accession GSM4687001 SRX8811081 GSM4687002 SRP273093 PRJNA647773 SRS7075127 GSM4687002 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687002 GSM4687002 GEO Accession GSM4687002 SRX8811082 GSM4687003 SRP273093 PRJNA647773 SRS7075126 GSM4687003 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687003 GSM4687003 GEO Accession GSM4687003 SRX8811083 GSM4687004 SRP273093 PRJNA647773 SRS7075128 GSM4687004 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687004 GSM4687004 GEO Accession GSM4687004 SRX8811084 GSM4687005 SRP273093 PRJNA647773 SRS7075129 GSM4687005 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687005 GSM4687005 GEO Accession GSM4687005 SRX8811085 GSM4687006 SRP273093 PRJNA647773 SRS7075130 GSM4687006 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687006 GSM4687006 GEO Accession GSM4687006 SRX8811086 GSM4687007 SRP273093 PRJNA647773 SRS7075131 GSM4687007 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687007 GSM4687007 GEO Accession GSM4687007 SRX8811087 GSM4687008 SRP273093 PRJNA647773 SRS7075132 GSM4687008 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687008 GSM4687008 GEO Accession GSM4687008 SRX8811088 GSM4687009 SRP273093 PRJNA647773 SRS7075133 GSM4687009 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687009 GSM4687009 GEO Accession GSM4687009 SRX8811089 GSM4687010 SRP273093 PRJNA647773 SRS7075134 GSM4687010 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687010 GSM4687010 GEO Accession GSM4687010 SRX8811090 GSM4687011 SRP273093 PRJNA647773 SRS7075135 GSM4687011 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687011 GSM4687011 GEO Accession GSM4687011 SRX8811091 GSM4687012 SRP273093 PRJNA647773 SRS7075136 GSM4687012 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687012 GSM4687012 GEO Accession GSM4687012 SRX8811092 GSM4687013 SRP273093 PRJNA647773 SRS7075137 GSM4687013 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687013 GSM4687013 GEO Accession GSM4687013 SRX8811093 GSM4687014 SRP273093 PRJNA647773 SRS7075138 GSM4687014 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687014 GSM4687014 GEO Accession GSM4687014 SRX8811094 GSM4687015 SRP273093 PRJNA647773 SRS7075139 GSM4687015 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687015 GSM4687015 GEO Accession GSM4687015 SRX8811095 GSM4687016 SRP273093 PRJNA647773 SRS7075140 GSM4687016 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687016 GSM4687016 GEO Accession GSM4687016 SRX8811096 GSM4687017 SRP273093 PRJNA647773 SRS7075141 GSM4687017 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687017 GSM4687017 GEO Accession GSM4687017 SRX8811097 GSM4687018 SRP273093 PRJNA647773 SRS7075142 GSM4687018 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687018 GSM4687018 GEO Accession GSM4687018 SRX8811098 GSM4687019 SRP273093 PRJNA647773 SRS7075144 GSM4687019 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687019 GSM4687019 GEO Accession GSM4687019 SRX8811099 GSM4687020 SRP273093 PRJNA647773 SRS7075143 GSM4687020 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687020 GSM4687020 GEO Accession GSM4687020 SRX8811100 GSM4687021 SRP273093 PRJNA647773 SRS7075145 GSM4687021 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687021 GSM4687021 GEO Accession GSM4687021 SRX8811101 GSM4687022 SRP273093 PRJNA647773 SRS7075146 GSM4687022 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687022 GSM4687022 GEO Accession GSM4687022 SRX8811102 GSM4687023 SRP273093 PRJNA647773 SRS7075147 GSM4687023 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687023 GSM4687023 GEO Accession GSM4687023 SRX8811103 GSM4687024 SRP273093 PRJNA647773 SRS7075148 GSM4687024 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687024 GSM4687024 GEO Accession GSM4687024 SRX8811105 GSM4687026 SRP273093 PRJNA647773 SRS7075150 GSM4687026 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687026 GSM4687026 GEO Accession GSM4687026 SRX8811106 GSM4687027 SRP273093 PRJNA647773 SRS7075151 GSM4687027 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687027 GSM4687027 GEO Accession GSM4687027 SRX8811107 GSM4687028 SRP273093 PRJNA647773 SRS7075152 GSM4687028 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687028 GSM4687028 GEO Accession GSM4687028 SRX8811108 GSM4687029 SRP273093 PRJNA647773 SRS7075153 GSM4687029 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687029 GSM4687029 GEO Accession GSM4687029 SRX8811109 GSM4687030 SRP273093 PRJNA647773 SRS7075154 GSM4687030 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687030 GSM4687030 GEO Accession GSM4687030 SRX8811110 GSM4687031 SRP273093 PRJNA647773 SRS7075155 GSM4687031 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687031 GSM4687031 GEO Accession GSM4687031 SRX8811111 GSM4687032 SRP273093 PRJNA647773 SRS7075156 GSM4687032 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687032 GSM4687032 GEO Accession GSM4687032 SRX8811112 GSM4687033 SRP273093 PRJNA647773 SRS7075157 GSM4687033 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687033 GSM4687033 GEO Accession GSM4687033 SRX8811113 GSM4687034 SRP273093 PRJNA647773 SRS7075158 GSM4687034 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687034 GSM4687034 GEO Accession GSM4687034 SRX8811114 GSM4687035 SRP273093 PRJNA647773 SRS7075159 GSM4687035 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687035 GSM4687035 GEO Accession GSM4687035 SRX8811115 GSM4687036 SRP273093 PRJNA647773 SRS7075160 GSM4687036 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687036 GSM4687036 GEO Accession GSM4687036 SRX8811116 GSM4687037 SRP273093 PRJNA647773 SRS7075162 GSM4687037 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687037 GSM4687037 GEO Accession GSM4687037 SRX8811117 GSM4687038 SRP273093 PRJNA647773 SRS7075161 GSM4687038 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687038 GSM4687038 GEO Accession GSM4687038 SRX8811118 GSM4687039 SRP273093 PRJNA647773 SRS7075163 GSM4687039 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687039 GSM4687039 GEO Accession GSM4687039 SRX8811119 GSM4687040 SRP273093 PRJNA647773 SRS7075164 GSM4687040 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687040 GSM4687040 GEO Accession GSM4687040 SRX8811120 GSM4687041 SRP273093 PRJNA647773 SRS7075165 GSM4687041 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687041 GSM4687041 GEO Accession GSM4687041 SRX8811121 GSM4687042 SRP273093 PRJNA647773 SRS7075166 GSM4687042 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687042 GSM4687042 GEO Accession GSM4687042 SRX8811122 GSM4687043 SRP273093 PRJNA647773 SRS7075167 GSM4687043 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687043 GSM4687043 GEO Accession GSM4687043 SRX8811123 GSM4687044 SRP273093 PRJNA647773 SRS7075168 GSM4687044 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687044 GSM4687044 GEO Accession GSM4687044 SRX8811124 GSM4687045 SRP273093 PRJNA647773 SRS7075169 GSM4687045 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687045 GSM4687045 GEO Accession GSM4687045 SRX8811125 GSM4687046 SRP273093 PRJNA647773 SRS7075170 GSM4687046 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687046 GSM4687046 GEO Accession GSM4687046 SRX8811126 GSM4687047 SRP273093 PRJNA647773 SRS7075171 GSM4687047 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687047 GSM4687047 GEO Accession GSM4687047 SRX8811127 GSM4687048 SRP273093 PRJNA647773 SRS7075172 GSM4687048 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687048 GSM4687048 GEO Accession GSM4687048 SRX8811128 GSM4687049 SRP273093 PRJNA647773 SRS7075173 GSM4687049 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687049 GSM4687049 GEO Accession GSM4687049 SRX8811129 GSM4687050 SRP273093 PRJNA647773 SRS7075175 GSM4687050 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687050 GSM4687050 GEO Accession GSM4687050 SRX8811130 GSM4687051 SRP273093 PRJNA647773 SRS7075174 GSM4687051 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687051 GSM4687051 GEO Accession GSM4687051 SRX8811131 GSM4687052 SRP273093 PRJNA647773 SRS7075176 GSM4687052 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687052 GSM4687052 GEO Accession GSM4687052 SRX8811132 GSM4687053 SRP273093 PRJNA647773 SRS7075177 GSM4687053 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687053 GSM4687053 GEO Accession GSM4687053 SRX8811133 GSM4687054 SRP273093 PRJNA647773 SRS7075178 GSM4687054 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687054 GSM4687054 GEO Accession GSM4687054 SRX8811134 GSM4687055 SRP273093 PRJNA647773 SRS7075179 GSM4687055 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687055 GSM4687055 GEO Accession GSM4687055 SRX8811135 GSM4687056 SRP273093 PRJNA647773 SRS7075180 GSM4687056 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687056 GSM4687056 GEO Accession GSM4687056 SRX8811136 GSM4687057 SRP273093 PRJNA647773 SRS7075181 GSM4687057 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687057 GSM4687057 GEO Accession GSM4687057 SRX8811137 GSM4687058 SRP273093 PRJNA647773 SRS7075182 GSM4687058 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687058 GSM4687058 GEO Accession GSM4687058 SRX8811138 GSM4687059 SRP273093 PRJNA647773 SRS7075183 GSM4687059 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687059 GSM4687059 GEO Accession GSM4687059 SRX8811139 GSM4687060 SRP273093 PRJNA647773 SRS7075184 GSM4687060 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687060 GSM4687060 GEO Accession GSM4687060 SRX8811140 GSM4687061 SRP273093 PRJNA647773 SRS7075185 GSM4687061 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687061 GSM4687061 GEO Accession GSM4687061 SRX8811141 GSM4687062 SRP273093 PRJNA647773 SRS7075186 GSM4687062 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687062 GSM4687062 GEO Accession GSM4687062 SRX8811142 GSM4687063 SRP273093 PRJNA647773 SRS7075187 GSM4687063 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687063 GSM4687063 GEO Accession GSM4687063 SRX8811143 GSM4687064 SRP273093 PRJNA647773 SRS7075188 GSM4687064 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687064 GSM4687064 GEO Accession GSM4687064 SRX8811144 GSM4687065 SRP273093 PRJNA647773 SRS7075189 GSM4687065 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687065 GSM4687065 GEO Accession GSM4687065 SRX8811145 GSM4687066 SRP273093 PRJNA647773 SRS7075191 GSM4687066 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687066 GSM4687066 GEO Accession GSM4687066 SRX8811146 GSM4687067 SRP273093 PRJNA647773 SRS7075190 GSM4687067 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687067 GSM4687067 GEO Accession GSM4687067 SRX8811147 GSM4687068 SRP273093 PRJNA647773 SRS7075192 GSM4687068 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687068 GSM4687068 GEO Accession GSM4687068 SRX8811148 GSM4687069 SRP273093 PRJNA647773 SRS7075193 GSM4687069 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687069 GSM4687069 GEO Accession GSM4687069 SRX8811149 GSM4687070 SRP273093 PRJNA647773 SRS7075194 GSM4687070 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687070 GSM4687070 GEO Accession GSM4687070 SRX8811150 GSM4687071 SRP273093 PRJNA647773 SRS7075195 GSM4687071 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687071 GSM4687071 GEO Accession GSM4687071 SRX8811151 GSM4687072 SRP273093 PRJNA647773 SRS7075196 GSM4687072 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687072 GSM4687072 GEO Accession GSM4687072 SRX8811152 GSM4687073 SRP273093 PRJNA647773 SRS7075197 GSM4687073 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687073 GSM4687073 GEO Accession GSM4687073 SRX8811153 GSM4687074 SRP273093 PRJNA647773 SRS7075199 GSM4687074 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687074 GSM4687074 GEO Accession GSM4687074 SRX8811154 GSM4687075 SRP273093 PRJNA647773 SRS7075198 GSM4687075 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687075 GSM4687075 GEO Accession GSM4687075 SRX8811155 GSM4687076 SRP273093 PRJNA647773 SRS7075200 GSM4687076 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687076 GSM4687076 GEO Accession GSM4687076 SRX8811156 GSM4687077 SRP273093 PRJNA647773 SRS7075202 GSM4687077 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687077 GSM4687077 GEO Accession GSM4687077 SRX8811157 GSM4687078 SRP273093 PRJNA647773 SRS7075201 GSM4687078 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687078 GSM4687078 GEO Accession GSM4687078 SRX8811158 GSM4687079 SRP273093 PRJNA647773 SRS7075203 GSM4687079 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687079 GSM4687079 GEO Accession GSM4687079 SRX8811159 GSM4687080 SRP273093 PRJNA647773 SRS7075204 GSM4687080 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687080 GSM4687080 GEO Accession GSM4687080 SRX8811160 GSM4687081 SRP273093 PRJNA647773 SRS7075205 GSM4687081 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687081 GSM4687081 GEO Accession GSM4687081 SRX8811161 GSM4687082 SRP273093 PRJNA647773 SRS7075206 GSM4687082 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687082 GSM4687082 GEO Accession GSM4687082 SRX8811162 GSM4687083 SRP273093 PRJNA647773 SRS7075207 GSM4687083 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687083 GSM4687083 GEO Accession GSM4687083 SRX8811163 GSM4687084 SRP273093 PRJNA647773 SRS7075208 GSM4687084 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687084 GSM4687084 GEO Accession GSM4687084 SRX8811164 GSM4687085 SRP273093 PRJNA647773 SRS7075209 GSM4687085 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687085 GSM4687085 GEO Accession GSM4687085 SRX8811165 GSM4687086 SRP273093 PRJNA647773 SRS7075210 GSM4687086 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687086 GSM4687086 GEO Accession GSM4687086 SRX8811166 GSM4687087 SRP273093 PRJNA647773 SRS7075211 GSM4687087 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687087 GSM4687087 GEO Accession GSM4687087 SRX8811167 GSM4687088 SRP273093 PRJNA647773 SRS7075212 GSM4687088 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687088 GSM4687088 GEO Accession GSM4687088 SRX8811168 GSM4687089 SRP273093 PRJNA647773 SRS7075213 GSM4687089 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687089 GSM4687089 GEO Accession GSM4687089 SRX8811169 GSM4687090 SRP273093 PRJNA647773 SRS7075214 GSM4687090 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687090 GSM4687090 GEO Accession GSM4687090 SRX8811170 GSM4687091 SRP273093 PRJNA647773 SRS7075216 GSM4687091 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687091 GSM4687091 GEO Accession GSM4687091 SRX8811171 GSM4687092 SRP273093 PRJNA647773 SRS7075215 GSM4687092 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687092 GSM4687092 GEO Accession GSM4687092 SRX8811172 GSM4687093 SRP273093 PRJNA647773 SRS7075217 GSM4687093 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687093 GSM4687093 GEO Accession GSM4687093 SRX8811173 GSM4687094 SRP273093 PRJNA647773 SRS7075218 GSM4687094 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687094 GSM4687094 GEO Accession GSM4687094 SRX8811174 GSM4687095 SRP273093 PRJNA647773 SRS7075219 GSM4687095 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687095 GSM4687095 GEO Accession GSM4687095 SRX8811175 GSM4687096 SRP273093 PRJNA647773 SRS7075220 GSM4687096 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687096 GSM4687096 GEO Accession GSM4687096 SRX8811176 GSM4687097 SRP273093 PRJNA647773 SRS7075221 GSM4687097 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687097 GSM4687097 GEO Accession GSM4687097 SRX8811177 GSM4687098 SRP273093 PRJNA647773 SRS7075222 GSM4687098 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687098 GSM4687098 GEO Accession GSM4687098 SRX8811178 GSM4687099 SRP273093 PRJNA647773 SRS7075223 GSM4687099 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687099 GSM4687099 GEO Accession GSM4687099 SRX8811179 GSM4687100 SRP273093 PRJNA647773 SRS7075224 GSM4687100 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687100 GSM4687100 GEO Accession GSM4687100 SRX8811180 GSM4687101 SRP273093 PRJNA647773 SRS7075622 GSM4687101 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687101 GSM4687101 GEO Accession GSM4687101 SRX8811181 GSM4687102 SRP273093 PRJNA647773 SRS7075623 GSM4687102 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687102 GSM4687102 GEO Accession GSM4687102 SRX8811182 GSM4687103 SRP273093 PRJNA647773 SRS7075624 GSM4687103 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687103 GSM4687103 GEO Accession GSM4687103 SRX8811183 GSM4687104 SRP273093 PRJNA647773 SRS7075625 GSM4687104 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687104 GSM4687104 GEO Accession GSM4687104 SRX8811184 GSM4687105 SRP273093 PRJNA647773 SRS7075626 GSM4687105 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687105 GSM4687105 GEO Accession GSM4687105 SRX8811185 GSM4687106 SRP273093 PRJNA647773 SRS7075628 GSM4687106 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687106 GSM4687106 GEO Accession GSM4687106 SRX8811186 GSM4687107 SRP273093 PRJNA647773 SRS7075627 GSM4687107 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687107 GSM4687107 GEO Accession GSM4687107 SRX8811187 GSM4687108 SRP273093 PRJNA647773 SRS7075629 GSM4687108 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687108 GSM4687108 GEO Accession GSM4687108 SRX8811188 GSM4687109 SRP273093 PRJNA647773 SRS7075630 GSM4687109 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687109 GSM4687109 GEO Accession GSM4687109 SRX8811189 GSM4687110 SRP273093 PRJNA647773 SRS7075631 GSM4687110 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687110 GSM4687110 GEO Accession GSM4687110 SRX8811190 GSM4687111 SRP273093 PRJNA647773 SRS7075632 GSM4687111 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687111 GSM4687111 GEO Accession GSM4687111 SRX8811191 GSM4687112 SRP273093 PRJNA647773 SRS7075633 GSM4687112 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687112 GSM4687112 GEO Accession GSM4687112 SRX8811192 GSM4687113 SRP273093 PRJNA647773 SRS7075635 GSM4687113 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687113 GSM4687113 GEO Accession GSM4687113 SRX8811193 GSM4687114 SRP273093 PRJNA647773 SRS7075634 GSM4687114 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687114 GSM4687114 GEO Accession GSM4687114 SRX8811194 GSM4687115 SRP273093 PRJNA647773 SRS7075636 GSM4687115 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687115 GSM4687115 GEO Accession GSM4687115 SRX8811195 GSM4687116 SRP273093 PRJNA647773 SRS7075637 GSM4687116 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687116 GSM4687116 GEO Accession GSM4687116 SRX8811196 GSM4687117 SRP273093 PRJNA647773 SRS7075638 GSM4687117 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687117 GSM4687117 GEO Accession GSM4687117 SRX8811197 GSM4687118 SRP273093 PRJNA647773 SRS7075639 GSM4687118 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687118 GSM4687118 GEO Accession GSM4687118 SRX8811198 GSM4687119 SRP273093 PRJNA647773 SRS7075640 GSM4687119 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687119 GSM4687119 GEO Accession GSM4687119 SRX8811199 GSM4687120 SRP273093 PRJNA647773 SRS7075641 GSM4687120 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687120 GSM4687120 GEO Accession GSM4687120 SRX8811200 GSM4687121 SRP273093 PRJNA647773 SRS7075642 GSM4687121 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687121 GSM4687121 GEO Accession GSM4687121 SRX8811201 GSM4687122 SRP273093 PRJNA647773 SRS7075643 GSM4687122 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687122 GSM4687122 GEO Accession GSM4687122 SRX8811202 GSM4687123 SRP273093 PRJNA647773 SRS7075644 GSM4687123 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687123 GSM4687123 GEO Accession GSM4687123 SRX8811203 GSM4687124 SRP273093 PRJNA647773 SRS7075645 GSM4687124 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687124 GSM4687124 GEO Accession GSM4687124 SRX8811204 GSM4687125 SRP273093 PRJNA647773 SRS7075646 GSM4687125 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687125 GSM4687125 GEO Accession GSM4687125 SRX8811205 GSM4687126 SRP273093 PRJNA647773 SRS7075647 GSM4687126 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687126 GSM4687126 GEO Accession GSM4687126 SRX8811206 GSM4687127 SRP273093 PRJNA647773 SRS7075648 GSM4687127 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687127 GSM4687127 GEO Accession GSM4687127 SRX8811207 GSM4687128 SRP273093 PRJNA647773 SRS7075649 GSM4687128 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687128 GSM4687128 GEO Accession GSM4687128 SRX8811208 GSM4687129 SRP273093 PRJNA647773 SRS7075650 GSM4687129 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687129 GSM4687129 GEO Accession GSM4687129 SRX8811209 GSM4687130 SRP273093 PRJNA647773 SRS7075651 GSM4687130 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687130 GSM4687130 GEO Accession GSM4687130 SRX8811210 GSM4687131 SRP273093 PRJNA647773 SRS7075225 GSM4687131 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687131 GSM4687131 GEO Accession GSM4687131 SRX8811211 GSM4687132 SRP273093 PRJNA647773 SRS7075226 GSM4687132 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687132 GSM4687132 GEO Accession GSM4687132 SRX8811212 GSM4687133 SRP273093 PRJNA647773 SRS7075227 GSM4687133 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687133 GSM4687133 GEO Accession GSM4687133 SRX8811213 GSM4687134 SRP273093 PRJNA647773 SRS7075228 GSM4687134 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687134 GSM4687134 GEO Accession GSM4687134 SRX8811214 GSM4687135 SRP273093 PRJNA647773 SRS7075229 GSM4687135 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687135 GSM4687135 GEO Accession GSM4687135 SRX8811215 GSM4687136 SRP273093 PRJNA647773 SRS7075230 GSM4687136 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687136 GSM4687136 GEO Accession GSM4687136 SRX8811216 GSM4687137 SRP273093 PRJNA647773 SRS7075231 GSM4687137 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687137 GSM4687137 GEO Accession GSM4687137 SRX8811217 GSM4687138 SRP273093 PRJNA647773 SRS7075232 GSM4687138 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687138 GSM4687138 GEO Accession GSM4687138 SRX8811218 GSM4687139 SRP273093 PRJNA647773 SRS7075233 GSM4687139 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687139 GSM4687139 GEO Accession GSM4687139 SRX8811219 GSM4687140 SRP273093 PRJNA647773 SRS7075234 GSM4687140 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687140 GSM4687140 GEO Accession GSM4687140 SRX8811220 GSM4687141 SRP273093 PRJNA647773 SRS7075235 GSM4687141 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687141 GSM4687141 GEO Accession GSM4687141 SRX8811221 GSM4687142 SRP273093 PRJNA647773 SRS7075236 GSM4687142 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687142 GSM4687142 GEO Accession GSM4687142 SRX8811222 GSM4687143 SRP273093 PRJNA647773 SRS7075237 GSM4687143 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687143 GSM4687143 GEO Accession GSM4687143 SRX8811223 GSM4687144 SRP273093 PRJNA647773 SRS7075238 GSM4687144 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687144 GSM4687144 GEO Accession GSM4687144 SRX8811224 GSM4687145 SRP273093 PRJNA647773 SRS7075239 GSM4687145 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687145 GSM4687145 GEO Accession GSM4687145 SRX8811225 GSM4687146 SRP273093 PRJNA647773 SRS7075240 GSM4687146 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687146 GSM4687146 GEO Accession GSM4687146 SRX8811226 GSM4687147 SRP273093 PRJNA647773 SRS7075241 GSM4687147 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687147 GSM4687147 GEO Accession GSM4687147 SRX8811227 GSM4687148 SRP273093 PRJNA647773 SRS7075242 GSM4687148 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687148 GSM4687148 GEO Accession GSM4687148 SRX8811228 GSM4687149 SRP273093 PRJNA647773 SRS7075243 GSM4687149 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687149 GSM4687149 GEO Accession GSM4687149 SRX8811229 GSM4687150 SRP273093 PRJNA647773 SRS7075244 GSM4687150 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687150 GSM4687150 GEO Accession GSM4687150 SRX8811230 GSM4687151 SRP273093 PRJNA647773 SRS7075245 GSM4687151 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687151 GSM4687151 GEO Accession GSM4687151 SRX8811231 GSM4687152 SRP273093 PRJNA647773 SRS7075246 GSM4687152 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687152 GSM4687152 GEO Accession GSM4687152 SRX8811232 GSM4687153 SRP273093 PRJNA647773 SRS7075247 GSM4687153 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687153 GSM4687153 GEO Accession GSM4687153 SRX8811233 GSM4687154 SRP273093 PRJNA647773 SRS7075652 GSM4687154 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687154 GSM4687154 GEO Accession GSM4687154 SRX8811234 GSM4687155 SRP273093 PRJNA647773 SRS7075653 GSM4687155 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687155 GSM4687155 GEO Accession GSM4687155 SRX8811235 GSM4687156 SRP273093 PRJNA647773 SRS7075654 GSM4687156 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687156 GSM4687156 GEO Accession GSM4687156 SRX8811236 GSM4687157 SRP273093 PRJNA647773 SRS7075655 GSM4687157 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687157 GSM4687157 GEO Accession GSM4687157 SRX8811237 GSM4687158 SRP273093 PRJNA647773 SRS7075656 GSM4687158 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687158 GSM4687158 GEO Accession GSM4687158 SRX8811238 GSM4687159 SRP273093 PRJNA647773 SRS7075657 GSM4687159 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687159 GSM4687159 GEO Accession GSM4687159 SRX8811239 GSM4687160 SRP273093 PRJNA647773 SRS7075658 GSM4687160 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687160 GSM4687160 GEO Accession GSM4687160 SRX8811240 GSM4687161 SRP273093 PRJNA647773 SRS7075248 GSM4687161 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687161 GSM4687161 GEO Accession GSM4687161 SRX8811241 GSM4687162 SRP273093 PRJNA647773 SRS7075250 GSM4687162 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687162 GSM4687162 GEO Accession GSM4687162 SRX8811242 GSM4687163 SRP273093 PRJNA647773 SRS7075249 GSM4687163 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687163 GSM4687163 GEO Accession GSM4687163 SRX8811243 GSM4687164 SRP273093 PRJNA647773 SRS7075251 GSM4687164 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687164 GSM4687164 GEO Accession GSM4687164 SRX8811244 GSM4687165 SRP273093 PRJNA647773 SRS7075252 GSM4687165 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687165 GSM4687165 GEO Accession GSM4687165 SRX8811245 GSM4687166 SRP273093 PRJNA647773 SRS7075253 GSM4687166 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687166 GSM4687166 GEO Accession GSM4687166 SRX8811246 GSM4687167 SRP273093 PRJNA647773 SRS7075254 GSM4687167 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687167 GSM4687167 GEO Accession GSM4687167 SRX8811247 GSM4687168 SRP273093 PRJNA647773 SRS7075255 GSM4687168 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687168 GSM4687168 GEO Accession GSM4687168 SRX8811248 GSM4687169 SRP273093 PRJNA647773 SRS7075257 GSM4687169 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687169 GSM4687169 GEO Accession GSM4687169 SRX8811249 GSM4687170 SRP273093 PRJNA647773 SRS7075256 GSM4687170 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687170 GSM4687170 GEO Accession GSM4687170 SRX8811250 GSM4687171 SRP273093 PRJNA647773 SRS7075258 GSM4687171 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687171 GSM4687171 GEO Accession GSM4687171 SRX8811251 GSM4687172 SRP273093 PRJNA647773 SRS7075259 GSM4687172 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687172 GSM4687172 GEO Accession GSM4687172 SRX8811252 GSM4687173 SRP273093 PRJNA647773 SRS7075260 GSM4687173 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687173 GSM4687173 GEO Accession GSM4687173 SRX8811253 GSM4687174 SRP273093 PRJNA647773 SRS7075262 GSM4687174 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687174 GSM4687174 GEO Accession GSM4687174 SRX8811254 GSM4687175 SRP273093 PRJNA647773 SRS7075261 GSM4687175 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687175 GSM4687175 GEO Accession GSM4687175 SRX8811255 GSM4687176 SRP273093 PRJNA647773 SRS7075263 GSM4687176 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687176 GSM4687176 GEO Accession GSM4687176 SRX8811256 GSM4687177 SRP273093 PRJNA647773 SRS7075264 GSM4687177 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687177 GSM4687177 GEO Accession GSM4687177 SRX8811257 GSM4687178 SRP273093 PRJNA647773 SRS7075265 GSM4687178 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687178 GSM4687178 GEO Accession GSM4687178 SRX8811258 GSM4687179 SRP273093 PRJNA647773 SRS7075267 GSM4687179 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687179 GSM4687179 GEO Accession GSM4687179 SRX8811259 GSM4687180 SRP273093 PRJNA647773 SRS7075266 GSM4687180 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687180 GSM4687180 GEO Accession GSM4687180 SRX8811260 GSM4687181 SRP273093 PRJNA647773 SRS7075268 GSM4687181 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687181 GSM4687181 GEO Accession GSM4687181 SRX8811261 GSM4687182 SRP273093 PRJNA647773 SRS7075269 GSM4687182 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687182 GSM4687182 GEO Accession GSM4687182 SRX8811262 GSM4687183 SRP273093 PRJNA647773 SRS7075270 GSM4687183 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687183 GSM4687183 GEO Accession GSM4687183 SRX8811263 GSM4687184 SRP273093 PRJNA647773 SRS7075271 GSM4687184 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687184 GSM4687184 GEO Accession GSM4687184 SRX8811264 GSM4687185 SRP273093 PRJNA647773 SRS7075272 GSM4687185 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687185 GSM4687185 GEO Accession GSM4687185 SRX8811265 GSM4687186 SRP273093 PRJNA647773 SRS7075273 GSM4687186 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687186 GSM4687186 GEO Accession GSM4687186 SRX8811266 GSM4687187 SRP273093 PRJNA647773 SRS7075274 GSM4687187 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687187 GSM4687187 GEO Accession GSM4687187 SRX8811267 GSM4687188 SRP273093 PRJNA647773 SRS7075275 GSM4687188 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687188 GSM4687188 GEO Accession GSM4687188 SRX8811268 GSM4687189 SRP273093 PRJNA647773 SRS7075277 GSM4687189 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687189 GSM4687189 GEO Accession GSM4687189 SRX8811269 GSM4687190 SRP273093 PRJNA647773 SRS7075276 GSM4687190 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687190 GSM4687190 GEO Accession GSM4687190 SRX8811270 GSM4687191 SRP273093 PRJNA647773 SRS7075659 GSM4687191 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687191 GSM4687191 GEO Accession GSM4687191 SRX8811271 GSM4687192 SRP273093 PRJNA647773 SRS7075660 GSM4687192 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687192 GSM4687192 GEO Accession GSM4687192 SRX8811272 GSM4687193 SRP273093 PRJNA647773 SRS7075662 GSM4687193 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687193 GSM4687193 GEO Accession GSM4687193 SRX8811273 GSM4687194 SRP273093 PRJNA647773 SRS7075661 GSM4687194 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687194 GSM4687194 GEO Accession GSM4687194 SRX8811274 GSM4687195 SRP273093 PRJNA647773 SRS7075663 GSM4687195 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687195 GSM4687195 GEO Accession GSM4687195 SRX8811275 GSM4687196 SRP273093 PRJNA647773 SRS7075664 GSM4687196 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687196 GSM4687196 GEO Accession GSM4687196 SRX8811276 GSM4687197 SRP273093 PRJNA647773 SRS7075665 GSM4687197 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687197 GSM4687197 GEO Accession GSM4687197 SRX8811277 GSM4687198 SRP273093 PRJNA647773 SRS7075666 GSM4687198 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687198 GSM4687198 GEO Accession GSM4687198 SRX8811278 GSM4687199 SRP273093 PRJNA647773 SRS7075667 GSM4687199 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687199 GSM4687199 GEO Accession GSM4687199 SRX8811279 GSM4687200 SRP273093 PRJNA647773 SRS7075668 GSM4687200 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687200 GSM4687200 GEO Accession GSM4687200 SRX8811280 GSM4687201 SRP273093 PRJNA647773 SRS7075669 GSM4687201 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687201 GSM4687201 GEO Accession GSM4687201 SRX8811281 GSM4687202 SRP273093 PRJNA647773 SRS7075670 GSM4687202 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687202 GSM4687202 GEO Accession GSM4687202 SRX8811282 GSM4687203 SRP273093 PRJNA647773 SRS7075671 GSM4687203 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687203 GSM4687203 GEO Accession GSM4687203 SRX8811283 GSM4687204 SRP273093 PRJNA647773 SRS7075672 GSM4687204 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687204 GSM4687204 GEO Accession GSM4687204 SRX8811284 GSM4687205 SRP273093 PRJNA647773 SRS7075673 GSM4687205 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687205 GSM4687205 GEO Accession GSM4687205 SRX8811285 GSM4687206 SRP273093 PRJNA647773 SRS7075674 GSM4687206 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687206 GSM4687206 GEO Accession GSM4687206 SRX8811286 GSM4687207 SRP273093 PRJNA647773 SRS7075675 GSM4687207 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687207 GSM4687207 GEO Accession GSM4687207 SRX8811287 GSM4687208 SRP273093 PRJNA647773 SRS7075676 GSM4687208 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687208 GSM4687208 GEO Accession GSM4687208 SRX8811288 GSM4687209 SRP273093 PRJNA647773 SRS7075677 GSM4687209 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687209 GSM4687209 GEO Accession GSM4687209 SRX8811289 GSM4687210 SRP273093 PRJNA647773 SRS7075678 GSM4687210 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687210 GSM4687210 GEO Accession GSM4687210 SRX8811290 GSM4687211 SRP273093 PRJNA647773 SRS7075679 GSM4687211 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687211 GSM4687211 GEO Accession GSM4687211 SRX8811291 GSM4687212 SRP273093 PRJNA647773 SRS7075681 GSM4687212 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687212 GSM4687212 GEO Accession GSM4687212 SRX8811292 GSM4687213 SRP273093 PRJNA647773 SRS7075680 GSM4687213 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687213 GSM4687213 GEO Accession GSM4687213 SRX8811293 GSM4687214 SRP273093 PRJNA647773 SRS7075682 GSM4687214 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687214 GSM4687214 GEO Accession GSM4687214 SRX8811294 GSM4687215 SRP273093 PRJNA647773 SRS7075683 GSM4687215 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687215 GSM4687215 GEO Accession GSM4687215 SRX8811295 GSM4687216 SRP273093 PRJNA647773 SRS7075684 GSM4687216 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687216 GSM4687216 GEO Accession GSM4687216 SRX8811296 GSM4687217 SRP273093 PRJNA647773 SRS7075685 GSM4687217 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687217 GSM4687217 GEO Accession GSM4687217 SRX8811297 GSM4687218 SRP273093 PRJNA647773 SRS7075686 GSM4687218 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687218 GSM4687218 GEO Accession GSM4687218 SRX8811298 GSM4687219 SRP273093 PRJNA647773 SRS7075687 GSM4687219 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687219 GSM4687219 GEO Accession GSM4687219 SRX8811299 GSM4687220 SRP273093 PRJNA647773 SRS7075688 GSM4687220 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687220 GSM4687220 GEO Accession GSM4687220 SRX8811300 GSM4687221 SRP273093 PRJNA647773 SRS7075689 GSM4687221 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687221 GSM4687221 GEO Accession GSM4687221 SRX8811301 GSM4687222 SRP273093 PRJNA647773 SRS7075690 GSM4687222 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687222 GSM4687222 GEO Accession GSM4687222 SRX8811302 GSM4687223 SRP273093 PRJNA647773 SRS7075691 GSM4687223 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687223 GSM4687223 GEO Accession GSM4687223 SRX8811303 GSM4687224 SRP273093 PRJNA647773 SRS7075692 GSM4687224 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687224 GSM4687224 GEO Accession GSM4687224 SRX8811304 GSM4687225 SRP273093 PRJNA647773 SRS7075693 GSM4687225 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687225 GSM4687225 GEO Accession GSM4687225 SRX8811305 GSM4687226 SRP273093 PRJNA647773 SRS7075694 GSM4687226 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687226 GSM4687226 GEO Accession GSM4687226 SRX8811306 GSM4687227 SRP273093 PRJNA647773 SRS7075695 GSM4687227 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687227 GSM4687227 GEO Accession GSM4687227 SRX8811307 GSM4687228 SRP273093 PRJNA647773 SRS7075696 GSM4687228 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687228 GSM4687228 GEO Accession GSM4687228 SRX8811308 GSM4687229 SRP273093 PRJNA647773 SRS7075697 GSM4687229 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687229 GSM4687229 GEO Accession GSM4687229 SRX8811309 GSM4687230 SRP273093 PRJNA647773 SRS7075698 GSM4687230 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687230 GSM4687230 GEO Accession GSM4687230 SRX8811310 GSM4687231 SRP273093 PRJNA647773 SRS7075699 GSM4687231 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687231 GSM4687231 GEO Accession GSM4687231 SRX8811311 GSM4687232 SRP273093 PRJNA647773 SRS7075700 GSM4687232 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687232 GSM4687232 GEO Accession GSM4687232 SRX8811312 GSM4687233 SRP273093 PRJNA647773 SRS7075701 GSM4687233 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687233 GSM4687233 GEO Accession GSM4687233 SRX8811313 GSM4687234 SRP273093 PRJNA647773 SRS7075702 GSM4687234 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687234 GSM4687234 GEO Accession GSM4687234 SRX8811314 GSM4687235 SRP273093 PRJNA647773 SRS7076224 GSM4687235 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687235 GSM4687235 GEO Accession GSM4687235 SRX8811315 GSM4687236 SRP273093 PRJNA647773 SRS7075703 GSM4687236 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687236 GSM4687236 GEO Accession GSM4687236 SRX8811316 GSM4687237 SRP273093 PRJNA647773 SRS7076225 GSM4687237 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687237 GSM4687237 GEO Accession GSM4687237 SRX8811317 GSM4687238 SRP273093 PRJNA647773 SRS7076226 GSM4687238 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687238 GSM4687238 GEO Accession GSM4687238 SRX8811318 GSM4687239 SRP273093 PRJNA647773 SRS7076227 GSM4687239 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687239 GSM4687239 GEO Accession GSM4687239 SRX8811319 GSM4687240 SRP273093 PRJNA647773 SRS7076229 GSM4687240 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687240 GSM4687240 GEO Accession GSM4687240 SRX8811320 GSM4687241 SRP273093 PRJNA647773 SRS7076228 GSM4687241 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687241 GSM4687241 GEO Accession GSM4687241 SRX8811321 GSM4687242 SRP273093 PRJNA647773 SRS7076230 GSM4687242 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687242 GSM4687242 GEO Accession GSM4687242 SRX8811322 GSM4687243 SRP273093 PRJNA647773 SRS7076231 GSM4687243 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687243 GSM4687243 GEO Accession GSM4687243 SRX8811323 GSM4687244 SRP273093 PRJNA647773 SRS7076233 GSM4687244 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687244 GSM4687244 GEO Accession GSM4687244 SRX8811324 GSM4687245 SRP273093 PRJNA647773 SRS7076232 GSM4687245 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687245 GSM4687245 GEO Accession GSM4687245 SRX8811325 GSM4687246 SRP273093 PRJNA647773 SRS7076234 GSM4687246 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687246 GSM4687246 GEO Accession GSM4687246 SRX8811326 GSM4687247 SRP273093 PRJNA647773 SRS7076235 GSM4687247 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687247 GSM4687247 GEO Accession GSM4687247 SRX8811327 GSM4687248 SRP273093 PRJNA647773 SRS7076236 GSM4687248 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687248 GSM4687248 GEO Accession GSM4687248 SRX8811328 GSM4687249 SRP273093 PRJNA647773 SRS7076237 GSM4687249 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687249 GSM4687249 GEO Accession GSM4687249 SRX8811329 GSM4687250 SRP273093 PRJNA647773 SRS7076238 GSM4687250 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687250 GSM4687250 GEO Accession GSM4687250 SRX8811330 GSM4687251 SRP273093 PRJNA647773 SRS7076240 GSM4687251 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687251 GSM4687251 GEO Accession GSM4687251 SRX8811331 GSM4687252 SRP273093 PRJNA647773 SRS7076239 GSM4687252 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687252 GSM4687252 GEO Accession GSM4687252 SRX8811332 GSM4687253 SRP273093 PRJNA647773 SRS7076241 GSM4687253 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687253 GSM4687253 GEO Accession GSM4687253 SRX8811333 GSM4687254 SRP273093 PRJNA647773 SRS7076242 GSM4687254 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687254 GSM4687254 GEO Accession GSM4687254 SRX8811334 GSM4687255 SRP273093 PRJNA647773 SRS7076243 GSM4687255 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687255 GSM4687255 GEO Accession GSM4687255 SRX8811335 GSM4687256 SRP273093 PRJNA647773 SRS7076244 GSM4687256 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687256 GSM4687256 GEO Accession GSM4687256 SRX8811336 GSM4687257 SRP273093 PRJNA647773 SRS7076245 GSM4687257 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687257 GSM4687257 GEO Accession GSM4687257 SRX8811337 GSM4687258 SRP273093 PRJNA647773 SRS7076246 GSM4687258 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687258 GSM4687258 GEO Accession GSM4687258 SRX8811338 GSM4687259 SRP273093 PRJNA647773 SRS7076247 GSM4687259 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687259 GSM4687259 GEO Accession GSM4687259 SRX8811339 GSM4687260 SRP273093 PRJNA647773 SRS7076248 GSM4687260 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687260 GSM4687260 GEO Accession GSM4687260 SRX8811340 GSM4687261 SRP273093 PRJNA647773 SRS7076249 GSM4687261 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687261 GSM4687261 GEO Accession GSM4687261 SRX8811341 GSM4687262 SRP273093 PRJNA647773 SRS7076250 GSM4687262 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687262 GSM4687262 GEO Accession GSM4687262 SRX8811342 GSM4687263 SRP273093 PRJNA647773 SRS7076252 GSM4687263 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687263 GSM4687263 GEO Accession GSM4687263 SRX8811343 GSM4687264 SRP273093 PRJNA647773 SRS7076251 GSM4687264 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687264 GSM4687264 GEO Accession GSM4687264 SRX8811344 GSM4687265 SRP273093 PRJNA647773 SRS7076253 GSM4687265 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687265 GSM4687265 GEO Accession GSM4687265 SRX8811345 GSM4687266 SRP273093 PRJNA647773 SRS7076255 GSM4687266 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687266 GSM4687266 GEO Accession GSM4687266 SRX8811346 GSM4687267 SRP273093 PRJNA647773 SRS7076254 GSM4687267 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687267 GSM4687267 GEO Accession GSM4687267 SRX8811347 GSM4687268 SRP273093 PRJNA647773 SRS7076256 GSM4687268 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687268 GSM4687268 GEO Accession GSM4687268 SRX8811348 GSM4687269 SRP273093 PRJNA647773 SRS7076257 GSM4687269 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687269 GSM4687269 GEO Accession GSM4687269 SRX8811349 GSM4687270 SRP273093 PRJNA647773 SRS7076258 GSM4687270 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687270 GSM4687270 GEO Accession GSM4687270 SRX8811350 GSM4687271 SRP273093 PRJNA647773 SRS7076259 GSM4687271 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687271 GSM4687271 GEO Accession GSM4687271 SRX8811351 GSM4687272 SRP273093 PRJNA647773 SRS7076260 GSM4687272 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687272 GSM4687272 GEO Accession GSM4687272 SRX8811352 GSM4687273 SRP273093 PRJNA647773 SRS7076261 GSM4687273 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687273 GSM4687273 GEO Accession GSM4687273 SRX8811353 GSM4687274 SRP273093 PRJNA647773 SRS7076262 GSM4687274 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687274 GSM4687274 GEO Accession GSM4687274 SRX8811354 GSM4687275 SRP273093 PRJNA647773 SRS7076263 GSM4687275 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687275 GSM4687275 GEO Accession GSM4687275 SRX8811355 GSM4687276 SRP273093 PRJNA647773 SRS7076265 GSM4687276 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687276 GSM4687276 GEO Accession GSM4687276 SRX8811356 GSM4687277 SRP273093 PRJNA647773 SRS7076264 GSM4687277 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687277 GSM4687277 GEO Accession GSM4687277 SRX8811357 GSM4687278 SRP273093 PRJNA647773 SRS7076266 GSM4687278 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687278 GSM4687278 GEO Accession GSM4687278 SRX8811358 GSM4687279 SRP273093 PRJNA647773 SRS7076267 GSM4687279 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687279 GSM4687279 GEO Accession GSM4687279 SRX8811359 GSM4687280 SRP273093 PRJNA647773 SRS7076268 GSM4687280 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687280 GSM4687280 GEO Accession GSM4687280 SRX8811360 GSM4687281 SRP273093 PRJNA647773 SRS7076269 GSM4687281 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687281 GSM4687281 GEO Accession GSM4687281 SRX8811361 GSM4687282 SRP273093 PRJNA647773 SRS7076270 GSM4687282 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687282 GSM4687282 GEO Accession GSM4687282 SRX8811362 GSM4687283 SRP273093 PRJNA647773 SRS7076271 GSM4687283 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687283 GSM4687283 GEO Accession GSM4687283 SRX8811363 GSM4687284 SRP273093 PRJNA647773 SRS7076272 GSM4687284 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687284 GSM4687284 GEO Accession GSM4687284 SRX8811364 GSM4687285 SRP273093 PRJNA647773 SRS7076273 GSM4687285 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687285 GSM4687285 GEO Accession GSM4687285 SRX8811365 GSM4687286 SRP273093 PRJNA647773 SRS7076275 GSM4687286 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687286 GSM4687286 GEO Accession GSM4687286 SRX8811366 GSM4687287 SRP273093 PRJNA647773 SRS7076274 GSM4687287 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687287 GSM4687287 GEO Accession GSM4687287 SRX8811367 GSM4687288 SRP273093 PRJNA647773 SRS7076276 GSM4687288 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687288 GSM4687288 GEO Accession GSM4687288 SRX8811368 GSM4687289 SRP273093 PRJNA647773 SRS7076278 GSM4687289 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687289 GSM4687289 GEO Accession GSM4687289 SRX8811369 GSM4687290 SRP273093 PRJNA647773 SRS7076277 GSM4687290 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687290 GSM4687290 GEO Accession GSM4687290 SRX8811370 GSM4687291 SRP273093 PRJNA647773 SRS7076280 GSM4687291 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687291 GSM4687291 GEO Accession GSM4687291 SRX8811371 GSM4687292 SRP273093 PRJNA647773 SRS7076279 GSM4687292 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687292 GSM4687292 GEO Accession GSM4687292 SRX8811372 GSM4687293 SRP273093 PRJNA647773 SRS7076281 GSM4687293 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687293 GSM4687293 GEO Accession GSM4687293 SRX8811373 GSM4687294 SRP273093 PRJNA647773 SRS7076284 GSM4687294 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687294 GSM4687294 GEO Accession GSM4687294 SRX8811374 GSM4687295 SRP273093 PRJNA647773 SRS7076282 GSM4687295 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687295 GSM4687295 GEO Accession GSM4687295 SRX8811375 GSM4687296 SRP273093 PRJNA647773 SRS7076283 GSM4687296 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687296 GSM4687296 GEO Accession GSM4687296 SRX8811376 GSM4687297 SRP273093 PRJNA647773 SRS7076285 GSM4687297 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687297 GSM4687297 GEO Accession GSM4687297 SRX8811377 GSM4687298 SRP273093 PRJNA647773 SRS7076286 GSM4687298 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687298 GSM4687298 GEO Accession GSM4687298 SRX8811378 GSM4687299 SRP273093 PRJNA647773 SRS7076287 GSM4687299 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687299 GSM4687299 GEO Accession GSM4687299 SRX8811379 GSM4687300 SRP273093 PRJNA647773 SRS7076288 GSM4687300 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687300 GSM4687300 GEO Accession GSM4687300 SRX8811380 GSM4687301 SRP273093 PRJNA647773 SRS7076289 GSM4687301 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687301 GSM4687301 GEO Accession GSM4687301 SRX8811381 GSM4687302 SRP273093 PRJNA647773 SRS7076290 GSM4687302 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687302 GSM4687302 GEO Accession GSM4687302 SRX8811382 GSM4687303 SRP273093 PRJNA647773 SRS7076291 GSM4687303 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687303 GSM4687303 GEO Accession GSM4687303 SRX8811383 GSM4687304 SRP273093 PRJNA647773 SRS7076292 GSM4687304 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687304 GSM4687304 GEO Accession GSM4687304 SRX8811384 GSM4687305 SRP273093 PRJNA647773 SRS7076293 GSM4687305 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687305 GSM4687305 GEO Accession GSM4687305 SRX8811385 GSM4687306 SRP273093 PRJNA647773 SRS7076294 GSM4687306 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687306 GSM4687306 GEO Accession GSM4687306 SRX8811386 GSM4687307 SRP273093 PRJNA647773 SRS7076295 GSM4687307 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687307 GSM4687307 GEO Accession GSM4687307 SRX8811387 GSM4687308 SRP273093 PRJNA647773 SRS7076296 GSM4687308 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687308 GSM4687308 GEO Accession GSM4687308 SRX8811388 GSM4687309 SRP273093 PRJNA647773 SRS7076298 GSM4687309 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687309 GSM4687309 GEO Accession GSM4687309 SRX8811389 GSM4687310 SRP273093 PRJNA647773 SRS7076297 GSM4687310 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687310 GSM4687310 GEO Accession GSM4687310 SRX8811390 GSM4687311 SRP273093 PRJNA647773 SRS7076299 GSM4687311 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687311 GSM4687311 GEO Accession GSM4687311 SRX8811391 GSM4687312 SRP273093 PRJNA647773 SRS7076301 GSM4687312 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687312 GSM4687312 GEO Accession GSM4687312 SRX8811392 GSM4687313 SRP273093 PRJNA647773 SRS7076302 GSM4687313 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687313 GSM4687313 GEO Accession GSM4687313 SRX8811393 GSM4687314 SRP273093 PRJNA647773 SRS7076300 GSM4687314 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687314 GSM4687314 GEO Accession GSM4687314 SRX8811394 GSM4687315 SRP273093 PRJNA647773 SRS7076303 GSM4687315 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687315 GSM4687315 GEO Accession GSM4687315 SRX8811395 GSM4687316 SRP273093 PRJNA647773 SRS7076304 GSM4687316 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687316 GSM4687316 GEO Accession GSM4687316 SRX8811396 GSM4687317 SRP273093 PRJNA647773 SRS7076305 GSM4687317 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687317 GSM4687317 GEO Accession GSM4687317 SRX8811397 GSM4687318 SRP273093 PRJNA647773 SRS7076306 GSM4687318 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687318 GSM4687318 GEO Accession GSM4687318 SRX8811398 GSM4687319 SRP273093 PRJNA647773 SRS7076307 GSM4687319 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687319 GSM4687319 GEO Accession GSM4687319 SRX8811399 GSM4687320 SRP273093 PRJNA647773 SRS7076308 GSM4687320 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687320 GSM4687320 GEO Accession GSM4687320 SRX8811400 GSM4687321 SRP273093 PRJNA647773 SRS7076310 GSM4687321 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687321 GSM4687321 GEO Accession GSM4687321 SRX8811401 GSM4687322 SRP273093 PRJNA647773 SRS7076309 GSM4687322 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687322 GSM4687322 GEO Accession GSM4687322 SRX8811402 GSM4687323 SRP273093 PRJNA647773 SRS7076311 GSM4687323 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687323 GSM4687323 GEO Accession GSM4687323 SRX8811403 GSM4687324 SRP273093 PRJNA647773 SRS7076313 GSM4687324 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687324 GSM4687324 GEO Accession GSM4687324 SRX8811404 GSM4687325 SRP273093 PRJNA647773 SRS7076312 GSM4687325 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687325 GSM4687325 GEO Accession GSM4687325 SRX8811405 GSM4687326 SRP273093 PRJNA647773 SRS7076315 GSM4687326 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687326 GSM4687326 GEO Accession GSM4687326 SRX8811406 GSM4687327 SRP273093 PRJNA647773 SRS7076314 GSM4687327 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687327 GSM4687327 GEO Accession GSM4687327 SRX8811407 GSM4687328 SRP273093 PRJNA647773 SRS7076316 GSM4687328 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687328 GSM4687328 GEO Accession GSM4687328 SRX8811408 GSM4687329 SRP273093 PRJNA647773 SRS7076318 GSM4687329 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687329 GSM4687329 GEO Accession GSM4687329 SRX8811409 GSM4687330 SRP273093 PRJNA647773 SRS7076317 GSM4687330 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687330 GSM4687330 GEO Accession GSM4687330 SRX8811410 GSM4687331 SRP273093 PRJNA647773 SRS7076319 GSM4687331 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687331 GSM4687331 GEO Accession GSM4687331 SRX8811411 GSM4687332 SRP273093 PRJNA647773 SRS7076320 GSM4687332 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687332 GSM4687332 GEO Accession GSM4687332 SRX8811412 GSM4687333 SRP273093 PRJNA647773 SRS7076321 GSM4687333 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687333 GSM4687333 GEO Accession GSM4687333 SRX8811413 GSM4687334 SRP273093 PRJNA647773 SRS7076322 GSM4687334 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687334 GSM4687334 GEO Accession GSM4687334 SRX8811414 GSM4687335 SRP273093 PRJNA647773 SRS7076324 GSM4687335 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687335 GSM4687335 GEO Accession GSM4687335 SRX8811415 GSM4687336 SRP273093 PRJNA647773 SRS7076325 GSM4687336 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687336 GSM4687336 GEO Accession GSM4687336 SRX8811416 GSM4687337 SRP273093 PRJNA647773 SRS7076323 GSM4687337 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687337 GSM4687337 GEO Accession GSM4687337 SRX8811417 GSM4687338 SRP273093 PRJNA647773 SRS7076326 GSM4687338 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687338 GSM4687338 GEO Accession GSM4687338 SRX8811418 GSM4687339 SRP273093 PRJNA647773 SRS7076327 GSM4687339 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687339 GSM4687339 GEO Accession GSM4687339 SRX8811419 GSM4687340 SRP273093 PRJNA647773 SRS7076329 GSM4687340 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687340 GSM4687340 GEO Accession GSM4687340 SRX8811420 GSM4687341 SRP273093 PRJNA647773 SRS7076328 GSM4687341 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687341 GSM4687341 GEO Accession GSM4687341 SRX8811421 GSM4687342 SRP273093 PRJNA647773 SRS7076330 GSM4687342 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687342 GSM4687342 GEO Accession GSM4687342 SRX8811422 GSM4687343 SRP273093 PRJNA647773 SRS7076331 GSM4687343 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687343 GSM4687343 GEO Accession GSM4687343 SRX8811423 GSM4687344 SRP273093 PRJNA647773 SRS7076332 GSM4687344 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687344 GSM4687344 GEO Accession GSM4687344 SRX8811424 GSM4687345 SRP273093 PRJNA647773 SRS7076334 GSM4687345 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687345 GSM4687345 GEO Accession GSM4687345 SRX8811425 GSM4687346 SRP273093 PRJNA647773 SRS7076333 GSM4687346 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687346 GSM4687346 GEO Accession GSM4687346 SRX8811426 GSM4687347 SRP273093 PRJNA647773 SRS7076335 GSM4687347 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687347 GSM4687347 GEO Accession GSM4687347 SRX8811427 GSM4687348 SRP273093 PRJNA647773 SRS7076336 GSM4687348 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687348 GSM4687348 GEO Accession GSM4687348 SRX8811428 GSM4687349 SRP273093 PRJNA647773 SRS7076337 GSM4687349 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687349 GSM4687349 GEO Accession GSM4687349 SRX8811429 GSM4687350 SRP273093 PRJNA647773 SRS7076338 GSM4687350 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687350 GSM4687350 GEO Accession GSM4687350 SRX8811430 GSM4687351 SRP273093 PRJNA647773 SRS7076339 GSM4687351 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687351 GSM4687351 GEO Accession GSM4687351 SRX8811431 GSM4687352 SRP273093 PRJNA647773 SRS7076340 GSM4687352 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687352 GSM4687352 GEO Accession GSM4687352 SRX8811432 GSM4687353 SRP273093 PRJNA647773 SRS7076341 GSM4687353 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687353 GSM4687353 GEO Accession GSM4687353 SRX8811433 GSM4687354 SRP273093 PRJNA647773 SRS7076342 GSM4687354 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687354 GSM4687354 GEO Accession GSM4687354 SRX8811434 GSM4687355 SRP273093 PRJNA647773 SRS7076343 GSM4687355 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687355 GSM4687355 GEO Accession GSM4687355 SRX8811435 GSM4687356 SRP273093 PRJNA647773 SRS7076344 GSM4687356 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687356 GSM4687356 GEO Accession GSM4687356 SRX8811436 GSM4687357 SRP273093 PRJNA647773 SRS7076345 GSM4687357 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687357 GSM4687357 GEO Accession GSM4687357 SRX8811437 GSM4687358 SRP273093 PRJNA647773 SRS7076346 GSM4687358 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687358 GSM4687358 GEO Accession GSM4687358 SRX8811438 GSM4687359 SRP273093 PRJNA647773 SRS7076348 GSM4687359 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687359 GSM4687359 GEO Accession GSM4687359 SRX8811439 GSM4687360 SRP273093 PRJNA647773 SRS7076349 GSM4687360 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687360 GSM4687360 GEO Accession GSM4687360 SRX8811440 GSM4687361 SRP273093 PRJNA647773 SRS7076347 GSM4687361 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687361 GSM4687361 GEO Accession GSM4687361 SRX8811441 GSM4687362 SRP273093 PRJNA647773 SRS7076350 GSM4687362 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687362 GSM4687362 GEO Accession GSM4687362 SRX8811442 GSM4687363 SRP273093 PRJNA647773 SRS7076351 GSM4687363 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687363 GSM4687363 GEO Accession GSM4687363 SRX8811443 GSM4687364 SRP273093 PRJNA647773 SRS7076352 GSM4687364 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687364 GSM4687364 GEO Accession GSM4687364 SRX8811444 GSM4687365 SRP273093 PRJNA647773 SRS7076353 GSM4687365 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687365 GSM4687365 GEO Accession GSM4687365 SRX8811445 GSM4687366 SRP273093 PRJNA647773 SRS7076355 GSM4687366 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687366 GSM4687366 GEO Accession GSM4687366 SRX8811446 GSM4687367 SRP273093 PRJNA647773 SRS7076354 GSM4687367 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687367 GSM4687367 GEO Accession GSM4687367 SRX8811447 GSM4687368 SRP273093 PRJNA647773 SRS7076356 GSM4687368 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687368 GSM4687368 GEO Accession GSM4687368 SRX8811448 GSM4687369 SRP273093 PRJNA647773 SRS7076357 GSM4687369 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687369 GSM4687369 GEO Accession GSM4687369 SRX8811449 GSM4687370 SRP273093 PRJNA647773 SRS7076358 GSM4687370 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687370 GSM4687370 GEO Accession GSM4687370 SRX8811450 GSM4687371 SRP273093 PRJNA647773 SRS7076359 GSM4687371 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687371 GSM4687371 GEO Accession GSM4687371 SRX8811451 GSM4687372 SRP273093 PRJNA647773 SRS7076362 GSM4687372 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687372 GSM4687372 GEO Accession GSM4687372 SRX8811452 GSM4687373 SRP273093 PRJNA647773 SRS7076360 GSM4687373 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687373 GSM4687373 GEO Accession GSM4687373 SRX8811453 GSM4687374 SRP273093 PRJNA647773 SRS7076361 GSM4687374 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687374 GSM4687374 GEO Accession GSM4687374 SRX8811454 GSM4687375 SRP273093 PRJNA647773 SRS7076364 GSM4687375 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687375 GSM4687375 GEO Accession GSM4687375 SRX8811455 GSM4687376 SRP273093 PRJNA647773 SRS7076363 GSM4687376 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687376 GSM4687376 GEO Accession GSM4687376 SRX8811456 GSM4687377 SRP273093 PRJNA647773 SRS7076365 GSM4687377 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687377 GSM4687377 GEO Accession GSM4687377 SRX8811457 GSM4687378 SRP273093 PRJNA647773 SRS7076366 GSM4687378 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687378 GSM4687378 GEO Accession GSM4687378 SRX8811458 GSM4687379 SRP273093 PRJNA647773 SRS7076369 GSM4687379 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687379 GSM4687379 GEO Accession GSM4687379 SRX8811459 GSM4687380 SRP273093 PRJNA647773 SRS7076368 GSM4687380 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687380 GSM4687380 GEO Accession GSM4687380 SRX8811460 GSM4687381 SRP273093 PRJNA647773 SRS7076367 GSM4687381 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687381 GSM4687381 GEO Accession GSM4687381 SRX8811461 GSM4687382 SRP273093 PRJNA647773 SRS7076370 GSM4687382 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687382 GSM4687382 GEO Accession GSM4687382 SRX8811462 GSM4687383 SRP273093 PRJNA647773 SRS7076371 GSM4687383 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687383 GSM4687383 GEO Accession GSM4687383 SRX8811463 GSM4687384 SRP273093 PRJNA647773 SRS7076372 GSM4687384 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687384 GSM4687384 GEO Accession GSM4687384 SRX8811464 GSM4687385 SRP273093 PRJNA647773 SRS7076373 GSM4687385 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687385 GSM4687385 GEO Accession GSM4687385 SRX8811465 GSM4687386 SRP273093 PRJNA647773 SRS7076374 GSM4687386 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687386 GSM4687386 GEO Accession GSM4687386 SRX8811466 GSM4687387 SRP273093 PRJNA647773 SRS7076376 GSM4687387 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687387 GSM4687387 GEO Accession GSM4687387 SRX8811467 GSM4687388 SRP273093 PRJNA647773 SRS7076375 GSM4687388 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687388 GSM4687388 GEO Accession GSM4687388 SRX8811468 GSM4687389 SRP273093 PRJNA647773 SRS7076377 GSM4687389 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687389 GSM4687389 GEO Accession GSM4687389 SRX8811469 GSM4687390 SRP273093 PRJNA647773 SRS7076379 GSM4687390 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687390 GSM4687390 GEO Accession GSM4687390 SRX8811470 GSM4687391 SRP273093 PRJNA647773 SRS7076378 GSM4687391 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687391 GSM4687391 GEO Accession GSM4687391 SRX8811471 GSM4687392 SRP273093 PRJNA647773 SRS7076380 GSM4687392 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687392 GSM4687392 GEO Accession GSM4687392 SRX8811472 GSM4687393 SRP273093 PRJNA647773 SRS7076381 GSM4687393 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687393 GSM4687393 GEO Accession GSM4687393 SRX8811473 GSM4687394 SRP273093 PRJNA647773 SRS7076382 GSM4687394 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687394 GSM4687394 GEO Accession GSM4687394 SRX8811474 GSM4687395 SRP273093 PRJNA647773 SRS7076383 GSM4687395 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687395 GSM4687395 GEO Accession GSM4687395 SRX8811475 GSM4687396 SRP273093 PRJNA647773 SRS7076384 GSM4687396 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687396 GSM4687396 GEO Accession GSM4687396 SRX8811476 GSM4687397 SRP273093 PRJNA647773 SRS7076385 GSM4687397 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687397 GSM4687397 GEO Accession GSM4687397 SRX8811477 GSM4687398 SRP273093 PRJNA647773 SRS7076386 GSM4687398 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687398 GSM4687398 GEO Accession GSM4687398 SRX8811478 GSM4687399 SRP273093 PRJNA647773 SRS7076387 GSM4687399 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687399 GSM4687399 GEO Accession GSM4687399 SRX8811479 GSM4687400 SRP273093 PRJNA647773 SRS7076388 GSM4687400 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687400 GSM4687400 GEO Accession GSM4687400 SRX8811480 GSM4687401 SRP273093 PRJNA647773 SRS7076389 GSM4687401 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687401 GSM4687401 GEO Accession GSM4687401 SRX8811481 GSM4687402 SRP273093 PRJNA647773 SRS7076391 GSM4687402 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687402 GSM4687402 GEO Accession GSM4687402 SRX8811482 GSM4687403 SRP273093 PRJNA647773 SRS7076390 GSM4687403 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687403 GSM4687403 GEO Accession GSM4687403 SRX8811483 GSM4687404 SRP273093 PRJNA647773 SRS7076392 GSM4687404 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687404 GSM4687404 GEO Accession GSM4687404 SRX8811484 GSM4687405 SRP273093 PRJNA647773 SRS7076394 GSM4687405 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687405 GSM4687405 GEO Accession GSM4687405 SRX8811485 GSM4687406 SRP273093 PRJNA647773 SRS7076393 GSM4687406 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687406 GSM4687406 GEO Accession GSM4687406 SRX8811486 GSM4687407 SRP273093 PRJNA647773 SRS7076395 GSM4687407 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687407 GSM4687407 GEO Accession GSM4687407 SRX8811487 GSM4687408 SRP273093 PRJNA647773 SRS7076396 GSM4687408 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687408 GSM4687408 GEO Accession GSM4687408 SRX8811488 GSM4687409 SRP273093 PRJNA647773 SRS7076397 GSM4687409 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687409 GSM4687409 GEO Accession GSM4687409 SRX8811489 GSM4687410 SRP273093 PRJNA647773 SRS7076398 GSM4687410 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687410 GSM4687410 GEO Accession GSM4687410 SRX8811490 GSM4687411 SRP273093 PRJNA647773 SRS7076399 GSM4687411 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687411 GSM4687411 GEO Accession GSM4687411 SRX8811491 GSM4687412 SRP273093 PRJNA647773 SRS7076400 GSM4687412 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687412 GSM4687412 GEO Accession GSM4687412 SRX8811492 GSM4687413 SRP273093 PRJNA647773 SRS7076402 GSM4687413 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687413 GSM4687413 GEO Accession GSM4687413 SRX8811493 GSM4687414 SRP273093 PRJNA647773 SRS7076401 GSM4687414 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687414 GSM4687414 GEO Accession GSM4687414 SRX8811494 GSM4687415 SRP273093 PRJNA647773 SRS7076403 GSM4687415 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687415 GSM4687415 GEO Accession GSM4687415 SRX8811495 GSM4687416 SRP273093 PRJNA647773 SRS7076404 GSM4687416 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687416 GSM4687416 GEO Accession GSM4687416 SRX8811496 GSM4687417 SRP273093 PRJNA647773 SRS7076405 GSM4687417 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687417 GSM4687417 GEO Accession GSM4687417 SRX8811497 GSM4687418 SRP273093 PRJNA647773 SRS7076406 GSM4687418 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687418 GSM4687418 GEO Accession GSM4687418 SRX8811498 GSM4687419 SRP273093 PRJNA647773 SRS7076408 GSM4687419 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687419 GSM4687419 GEO Accession GSM4687419 SRX8811499 GSM4687420 SRP273093 PRJNA647773 SRS7076407 GSM4687420 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687420 GSM4687420 GEO Accession GSM4687420 SRX8811500 GSM4687421 SRP273093 PRJNA647773 SRS7076410 GSM4687421 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687421 GSM4687421 GEO Accession GSM4687421 SRX8811501 GSM4687422 SRP273093 PRJNA647773 SRS7076409 GSM4687422 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687422 GSM4687422 GEO Accession GSM4687422 SRX8811502 GSM4687423 SRP273093 PRJNA647773 SRS7076411 GSM4687423 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687423 GSM4687423 GEO Accession GSM4687423 SRX8811503 GSM4687424 SRP273093 PRJNA647773 SRS7076412 GSM4687424 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687424 GSM4687424 GEO Accession GSM4687424 SRX8811504 GSM4687425 SRP273093 PRJNA647773 SRS7076413 GSM4687425 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687425 GSM4687425 GEO Accession GSM4687425 SRX8811505 GSM4687426 SRP273093 PRJNA647773 SRS7076414 GSM4687426 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687426 GSM4687426 GEO Accession GSM4687426 SRX8811506 GSM4687427 SRP273093 PRJNA647773 SRS7076415 GSM4687427 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687427 GSM4687427 GEO Accession GSM4687427 SRX8811507 GSM4687428 SRP273093 PRJNA647773 SRS7076418 GSM4687428 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687428 GSM4687428 GEO Accession GSM4687428 SRX8811508 GSM4687429 SRP273093 PRJNA647773 SRS7076416 GSM4687429 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687429 GSM4687429 GEO Accession GSM4687429 SRX8811509 GSM4687430 SRP273093 PRJNA647773 SRS7076417 GSM4687430 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687430 GSM4687430 GEO Accession GSM4687430 SRX8811510 GSM4687431 SRP273093 PRJNA647773 SRS7076420 GSM4687431 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687431 GSM4687431 GEO Accession GSM4687431 SRX8811511 GSM4687432 SRP273093 PRJNA647773 SRS7076419 GSM4687432 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687432 GSM4687432 GEO Accession GSM4687432 SRX8811512 GSM4687433 SRP273093 PRJNA647773 SRS7076421 GSM4687433 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687433 GSM4687433 GEO Accession GSM4687433 SRX8811513 GSM4687434 SRP273093 PRJNA647773 SRS7076423 GSM4687434 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687434 GSM4687434 GEO Accession GSM4687434 SRX8811514 GSM4687435 SRP273093 PRJNA647773 SRS7076422 GSM4687435 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687435 GSM4687435 GEO Accession GSM4687435 SRX8811515 GSM4687436 SRP273093 PRJNA647773 SRS7076424 GSM4687436 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687436 GSM4687436 GEO Accession GSM4687436 SRX8811516 GSM4687437 SRP273093 PRJNA647773 SRS7076425 GSM4687437 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687437 GSM4687437 GEO Accession GSM4687437 SRX8811517 GSM4687438 SRP273093 PRJNA647773 SRS7076427 GSM4687438 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687438 GSM4687438 GEO Accession GSM4687438 SRX8811518 GSM4687439 SRP273093 PRJNA647773 SRS7076426 GSM4687439 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687439 GSM4687439 GEO Accession GSM4687439 SRX8811519 GSM4687440 SRP273093 PRJNA647773 SRS7076429 GSM4687440 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687440 GSM4687440 GEO Accession GSM4687440 SRX8811520 GSM4687441 SRP273093 PRJNA647773 SRS7076430 GSM4687441 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687441 GSM4687441 GEO Accession GSM4687441 SRX8811521 GSM4687442 SRP273093 PRJNA647773 SRS7076428 GSM4687442 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687442 GSM4687442 GEO Accession GSM4687442 SRX8811522 GSM4687443 SRP273093 PRJNA647773 SRS7076431 GSM4687443 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687443 GSM4687443 GEO Accession GSM4687443 SRX8811523 GSM4687444 SRP273093 PRJNA647773 SRS7076432 GSM4687444 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687444 GSM4687444 GEO Accession GSM4687444 SRX8811524 GSM4687445 SRP273093 PRJNA647773 SRS7076433 GSM4687445 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687445 GSM4687445 GEO Accession GSM4687445 SRX8811525 GSM4687446 SRP273093 PRJNA647773 SRS7076434 GSM4687446 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687446 GSM4687446 GEO Accession GSM4687446 SRX8811526 GSM4687447 SRP273093 PRJNA647773 SRS7076435 GSM4687447 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687447 GSM4687447 GEO Accession GSM4687447 SRX8811527 GSM4687448 SRP273093 PRJNA647773 SRS7076436 GSM4687448 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687448 GSM4687448 GEO Accession GSM4687448 SRX8811528 GSM4687449 SRP273093 PRJNA647773 SRS7076437 GSM4687449 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687449 GSM4687449 GEO Accession GSM4687449 SRX8811529 GSM4687450 SRP273093 PRJNA647773 SRS7076438 GSM4687450 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687450 GSM4687450 GEO Accession GSM4687450 SRX8811530 GSM4687451 SRP273093 PRJNA647773 SRS7076439 GSM4687451 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687451 GSM4687451 GEO Accession GSM4687451 SRX8811531 GSM4687452 SRP273093 PRJNA647773 SRS7076440 GSM4687452 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687452 GSM4687452 GEO Accession GSM4687452 SRX8811532 GSM4687453 SRP273093 PRJNA647773 SRS7076441 GSM4687453 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687453 GSM4687453 GEO Accession GSM4687453 SRX8811533 GSM4687454 SRP273093 PRJNA647773 SRS7076443 GSM4687454 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687454 GSM4687454 GEO Accession GSM4687454 SRX8811534 GSM4687455 SRP273093 PRJNA647773 SRS7076442 GSM4687455 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687455 GSM4687455 GEO Accession GSM4687455 SRX8811535 GSM4687456 SRP273093 PRJNA647773 SRS7076444 GSM4687456 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687456 GSM4687456 GEO Accession GSM4687456 SRX8811536 GSM4687457 SRP273093 PRJNA647773 SRS7076445 GSM4687457 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687457 GSM4687457 GEO Accession GSM4687457 SRX8811537 GSM4687458 SRP273093 PRJNA647773 SRS7076447 GSM4687458 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687458 GSM4687458 GEO Accession GSM4687458 SRX8811538 GSM4687459 SRP273093 PRJNA647773 SRS7076446 GSM4687459 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687459 GSM4687459 GEO Accession GSM4687459 SRX8811539 GSM4687460 SRP273093 PRJNA647773 SRS7076448 GSM4687460 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687460 GSM4687460 GEO Accession GSM4687460 SRX8811540 GSM4687461 SRP273093 PRJNA647773 SRS7076450 GSM4687461 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687461 GSM4687461 GEO Accession GSM4687461 SRX8811541 GSM4687462 SRP273093 PRJNA647773 SRS7076452 GSM4687462 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687462 GSM4687462 GEO Accession GSM4687462 SRX8811542 GSM4687463 SRP273093 PRJNA647773 SRS7076449 GSM4687463 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687463 GSM4687463 GEO Accession GSM4687463 SRX8811543 GSM4687464 SRP273093 PRJNA647773 SRS7076451 GSM4687464 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687464 GSM4687464 GEO Accession GSM4687464 SRX8811544 GSM4687465 SRP273093 PRJNA647773 SRS7076453 GSM4687465 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687465 GSM4687465 GEO Accession GSM4687465 SRX8811545 GSM4687466 SRP273093 PRJNA647773 SRS7076454 GSM4687466 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687466 GSM4687466 GEO Accession GSM4687466 SRX8811546 GSM4687467 SRP273093 PRJNA647773 SRS7076455 GSM4687467 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687467 GSM4687467 GEO Accession GSM4687467 SRX8811547 GSM4687468 SRP273093 PRJNA647773 SRS7076456 GSM4687468 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687468 GSM4687468 GEO Accession GSM4687468 SRX8811548 GSM4687469 SRP273093 PRJNA647773 SRS7076457 GSM4687469 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687469 GSM4687469 GEO Accession GSM4687469 SRX8811549 GSM4687470 SRP273093 PRJNA647773 SRS7076458 GSM4687470 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687470 GSM4687470 GEO Accession GSM4687470 SRX8811550 GSM4687471 SRP273093 PRJNA647773 SRS7076459 GSM4687471 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687471 GSM4687471 GEO Accession GSM4687471 SRX8811551 GSM4687472 SRP273093 PRJNA647773 SRS7076460 GSM4687472 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687472 GSM4687472 GEO Accession GSM4687472 SRX8811552 GSM4687473 SRP273093 PRJNA647773 SRS7076461 GSM4687473 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687473 GSM4687473 GEO Accession GSM4687473 SRX8811553 GSM4687474 SRP273093 PRJNA647773 SRS7076462 GSM4687474 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687474 GSM4687474 GEO Accession GSM4687474 SRX8811554 GSM4687475 SRP273093 PRJNA647773 SRS7076463 GSM4687475 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687475 GSM4687475 GEO Accession GSM4687475 SRX8811555 GSM4687476 SRP273093 PRJNA647773 SRS7076464 GSM4687476 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687476 GSM4687476 GEO Accession GSM4687476 SRX8811556 GSM4687477 SRP273093 PRJNA647773 SRS7076465 GSM4687477 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687477 GSM4687477 GEO Accession GSM4687477 SRX8811557 GSM4687478 SRP273093 PRJNA647773 SRS7076466 GSM4687478 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687478 GSM4687478 GEO Accession GSM4687478 SRX8811558 GSM4687479 SRP273093 PRJNA647773 SRS7076469 GSM4687479 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687479 GSM4687479 GEO Accession GSM4687479 SRX8811559 GSM4687480 SRP273093 PRJNA647773 SRS7076467 GSM4687480 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687480 GSM4687480 GEO Accession GSM4687480 SRX8811560 GSM4687481 SRP273093 PRJNA647773 SRS7076468 GSM4687481 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687481 GSM4687481 GEO Accession GSM4687481 SRX8811561 GSM4687482 SRP273093 PRJNA647773 SRS7076470 GSM4687482 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687482 GSM4687482 GEO Accession GSM4687482 SRX8811562 GSM4687483 SRP273093 PRJNA647773 SRS7076471 GSM4687483 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687483 GSM4687483 GEO Accession GSM4687483 SRX8811563 GSM4687484 SRP273093 PRJNA647773 SRS7076472 GSM4687484 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687484 GSM4687484 GEO Accession GSM4687484 SRX8811564 GSM4687485 SRP273093 PRJNA647773 SRS7076473 GSM4687485 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687485 GSM4687485 GEO Accession GSM4687485 SRX8811565 GSM4687486 SRP273093 PRJNA647773 SRS7076474 GSM4687486 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687486 GSM4687486 GEO Accession GSM4687486 SRX8811566 GSM4687487 SRP273093 PRJNA647773 SRS7076475 GSM4687487 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687487 GSM4687487 GEO Accession GSM4687487 SRX8811567 GSM4687488 SRP273093 PRJNA647773 SRS7076477 GSM4687488 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687488 GSM4687488 GEO Accession GSM4687488 SRX8811568 GSM4687489 SRP273093 PRJNA647773 SRS7076476 GSM4687489 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687489 GSM4687489 GEO Accession GSM4687489 SRX8811569 GSM4687490 SRP273093 PRJNA647773 SRS7076478 GSM4687490 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687490 GSM4687490 GEO Accession GSM4687490 SRX8811570 GSM4687491 SRP273093 PRJNA647773 SRS7076480 GSM4687491 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687491 GSM4687491 GEO Accession GSM4687491 SRX8811571 GSM4687492 SRP273093 PRJNA647773 SRS7076479 GSM4687492 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687492 GSM4687492 GEO Accession GSM4687492 SRX8811572 GSM4687493 SRP273093 PRJNA647773 SRS7076481 GSM4687493 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687493 GSM4687493 GEO Accession GSM4687493 SRX8811573 GSM4687494 SRP273093 PRJNA647773 SRS7076482 GSM4687494 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687494 GSM4687494 GEO Accession GSM4687494 SRX8811574 GSM4687495 SRP273093 PRJNA647773 SRS7076483 GSM4687495 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687495 GSM4687495 GEO Accession GSM4687495 SRX8811575 GSM4687496 SRP273093 PRJNA647773 SRS7076485 GSM4687496 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687496 GSM4687496 GEO Accession GSM4687496 SRX8811576 GSM4687497 SRP273093 PRJNA647773 SRS7076484 GSM4687497 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687497 GSM4687497 GEO Accession GSM4687497 SRX8811577 GSM4687498 SRP273093 PRJNA647773 SRS7076486 GSM4687498 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687498 GSM4687498 GEO Accession GSM4687498 SRX8811578 GSM4687499 SRP273093 PRJNA647773 SRS7076487 GSM4687499 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687499 GSM4687499 GEO Accession GSM4687499 SRX8811579 GSM4687500 SRP273093 PRJNA647773 SRS7076489 GSM4687500 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687500 GSM4687500 GEO Accession GSM4687500 SRX8811580 GSM4687501 SRP273093 PRJNA647773 SRS7076488 GSM4687501 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687501 GSM4687501 GEO Accession GSM4687501 SRX8811581 GSM4687502 SRP273093 PRJNA647773 SRS7076490 GSM4687502 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687502 GSM4687502 GEO Accession GSM4687502 SRX8811582 GSM4687503 SRP273093 PRJNA647773 SRS7076491 GSM4687503 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687503 GSM4687503 GEO Accession GSM4687503 SRX8811583 GSM4687504 SRP273093 PRJNA647773 SRS7076493 GSM4687504 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687504 GSM4687504 GEO Accession GSM4687504 SRX8811584 GSM4687505 SRP273093 PRJNA647773 SRS7076492 GSM4687505 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687505 GSM4687505 GEO Accession GSM4687505 SRX8811585 GSM4687506 SRP273093 PRJNA647773 SRS7076495 GSM4687506 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687506 GSM4687506 GEO Accession GSM4687506 SRX8811586 GSM4687507 SRP273093 PRJNA647773 SRS7076494 GSM4687507 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687507 GSM4687507 GEO Accession GSM4687507 SRX8811587 GSM4687508 SRP273093 PRJNA647773 SRS7076496 GSM4687508 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687508 GSM4687508 GEO Accession GSM4687508 SRX8811588 GSM4687509 SRP273093 PRJNA647773 SRS7076497 GSM4687509 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687509 GSM4687509 GEO Accession GSM4687509 SRX8811589 GSM4687510 SRP273093 PRJNA647773 SRS7076498 GSM4687510 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687510 GSM4687510 GEO Accession GSM4687510 SRX8811590 GSM4687511 SRP273093 PRJNA647773 SRS7076499 GSM4687511 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687511 GSM4687511 GEO Accession GSM4687511 SRX8811591 GSM4687512 SRP273093 PRJNA647773 SRS7076501 GSM4687512 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687512 GSM4687512 GEO Accession GSM4687512 SRX8811592 GSM4687513 SRP273093 PRJNA647773 SRS7076500 GSM4687513 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687513 GSM4687513 GEO Accession GSM4687513 SRX8811593 GSM4687514 SRP273093 PRJNA647773 SRS7076502 GSM4687514 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687514 GSM4687514 GEO Accession GSM4687514 SRX8811594 GSM4687515 SRP273093 PRJNA647773 SRS7076503 GSM4687515 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687515 GSM4687515 GEO Accession GSM4687515 SRX8811595 GSM4687516 SRP273093 PRJNA647773 SRS7076506 GSM4687516 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687516 GSM4687516 GEO Accession GSM4687516 SRX8811596 GSM4687517 SRP273093 PRJNA647773 SRS7076504 GSM4687517 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687517 GSM4687517 GEO Accession GSM4687517 SRX8811597 GSM4687518 SRP273093 PRJNA647773 SRS7076505 GSM4687518 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687518 GSM4687518 GEO Accession GSM4687518 SRX8811598 GSM4687519 SRP273093 PRJNA647773 SRS7076509 GSM4687519 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687519 GSM4687519 GEO Accession GSM4687519 SRX8811599 GSM4687520 SRP273093 PRJNA647773 SRS7076507 GSM4687520 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687520 GSM4687520 GEO Accession GSM4687520 SRX8811600 GSM4687521 SRP273093 PRJNA647773 SRS7076508 GSM4687521 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687521 GSM4687521 GEO Accession GSM4687521 SRX8811601 GSM4687522 SRP273093 PRJNA647773 SRS7076510 GSM4687522 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687522 GSM4687522 GEO Accession GSM4687522 SRX8811602 GSM4687523 SRP273093 PRJNA647773 SRS7076511 GSM4687523 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687523 GSM4687523 GEO Accession GSM4687523 SRX8811603 GSM4687524 SRP273093 PRJNA647773 SRS7076512 GSM4687524 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687524 GSM4687524 GEO Accession GSM4687524 SRX8811604 GSM4687525 SRP273093 PRJNA647773 SRS7076513 GSM4687525 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687525 GSM4687525 GEO Accession GSM4687525 SRX8811605 GSM4687526 SRP273093 PRJNA647773 SRS7076514 GSM4687526 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687526 GSM4687526 GEO Accession GSM4687526 SRX8811606 GSM4687527 SRP273093 PRJNA647773 SRS7076515 GSM4687527 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687527 GSM4687527 GEO Accession GSM4687527 SRX8811607 GSM4687528 SRP273093 PRJNA647773 SRS7076516 GSM4687528 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687528 GSM4687528 GEO Accession GSM4687528 SRX8811608 GSM4687529 SRP273093 PRJNA647773 SRS7076517 GSM4687529 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687529 GSM4687529 GEO Accession GSM4687529 SRX8811609 GSM4687530 SRP273093 PRJNA647773 SRS7076519 GSM4687530 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687530 GSM4687530 GEO Accession GSM4687530 SRX8811610 GSM4687531 SRP273093 PRJNA647773 SRS7076518 GSM4687531 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687531 GSM4687531 GEO Accession GSM4687531 SRX8811611 GSM4687532 SRP273093 PRJNA647773 SRS7076520 GSM4687532 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687532 GSM4687532 GEO Accession GSM4687532 SRX8811612 GSM4687533 SRP273093 PRJNA647773 SRS7076521 GSM4687533 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687533 GSM4687533 GEO Accession GSM4687533 SRX8811613 GSM4687534 SRP273093 PRJNA647773 SRS7076523 GSM4687534 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687534 GSM4687534 GEO Accession GSM4687534 SRX8811614 GSM4687535 SRP273093 PRJNA647773 SRS7076522 GSM4687535 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687535 GSM4687535 GEO Accession GSM4687535 SRX8811615 GSM4687536 SRP273093 PRJNA647773 SRS7076524 GSM4687536 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687536 GSM4687536 GEO Accession GSM4687536 SRX8811616 GSM4687537 SRP273093 PRJNA647773 SRS7076525 GSM4687537 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687537 GSM4687537 GEO Accession GSM4687537 SRX8811617 GSM4687538 SRP273093 PRJNA647773 SRS7076526 GSM4687538 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687538 GSM4687538 GEO Accession GSM4687538 SRX8811618 GSM4687539 SRP273093 PRJNA647773 SRS7076527 GSM4687539 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687539 GSM4687539 GEO Accession GSM4687539 SRX8811619 GSM4687540 SRP273093 PRJNA647773 SRS7076529 GSM4687540 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687540 GSM4687540 GEO Accession GSM4687540 SRX8811620 GSM4687541 SRP273093 PRJNA647773 SRS7076528 GSM4687541 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687541 GSM4687541 GEO Accession GSM4687541 SRX8811621 GSM4687542 SRP273093 PRJNA647773 SRS7076530 GSM4687542 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687542 GSM4687542 GEO Accession GSM4687542 SRX8811622 GSM4687543 SRP273093 PRJNA647773 SRS7076531 GSM4687543 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687543 GSM4687543 GEO Accession GSM4687543 SRX8811623 GSM4687544 SRP273093 PRJNA647773 SRS7076533 GSM4687544 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687544 GSM4687544 GEO Accession GSM4687544 SRX8811624 GSM4687545 SRP273093 PRJNA647773 SRS7076532 GSM4687545 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687545 GSM4687545 GEO Accession GSM4687545 SRX8811625 GSM4687546 SRP273093 PRJNA647773 SRS7076534 GSM4687546 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687546 GSM4687546 GEO Accession GSM4687546 SRX8811626 GSM4687547 SRP273093 PRJNA647773 SRS7076536 GSM4687547 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687547 GSM4687547 GEO Accession GSM4687547 SRX8811627 GSM4687548 SRP273093 PRJNA647773 SRS7076535 GSM4687548 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687548 GSM4687548 GEO Accession GSM4687548 SRX8811628 GSM4687549 SRP273093 PRJNA647773 SRS7076537 GSM4687549 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687549 GSM4687549 GEO Accession GSM4687549 SRX8811629 GSM4687550 SRP273093 PRJNA647773 SRS7076539 GSM4687550 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687550 GSM4687550 GEO Accession GSM4687550 SRX8811630 GSM4687551 SRP273093 PRJNA647773 SRS7076538 GSM4687551 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687551 GSM4687551 GEO Accession GSM4687551 SRX8811631 GSM4687552 SRP273093 PRJNA647773 SRS7076540 GSM4687552 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687552 GSM4687552 GEO Accession GSM4687552 SRX8811632 GSM4687553 SRP273093 PRJNA647773 SRS7076541 GSM4687553 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687553 GSM4687553 GEO Accession GSM4687553 SRX8811633 GSM4687554 SRP273093 PRJNA647773 SRS7076542 GSM4687554 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687554 GSM4687554 GEO Accession GSM4687554 SRX8811634 GSM4687555 SRP273093 PRJNA647773 SRS7076543 GSM4687555 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687555 GSM4687555 GEO Accession GSM4687555 SRX8811635 GSM4687556 SRP273093 PRJNA647773 SRS7076544 GSM4687556 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687556 GSM4687556 GEO Accession GSM4687556 SRX8811636 GSM4687557 SRP273093 PRJNA647773 SRS7076545 GSM4687557 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687557 GSM4687557 GEO Accession GSM4687557 SRX8811637 GSM4687558 SRP273093 PRJNA647773 SRS7076546 GSM4687558 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687558 GSM4687558 GEO Accession GSM4687558 SRX8811638 GSM4687559 SRP273093 PRJNA647773 SRS7076547 GSM4687559 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687559 GSM4687559 GEO Accession GSM4687559 SRX8811639 GSM4687560 SRP273093 PRJNA647773 SRS7076548 GSM4687560 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687560 GSM4687560 GEO Accession GSM4687560 SRX8811640 GSM4687561 SRP273093 PRJNA647773 SRS7076549 GSM4687561 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687561 GSM4687561 GEO Accession GSM4687561 SRX8811641 GSM4687562 SRP273093 PRJNA647773 SRS7076550 GSM4687562 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687562 GSM4687562 GEO Accession GSM4687562 SRX8811642 GSM4687563 SRP273093 PRJNA647773 SRS7076551 GSM4687563 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687563 GSM4687563 GEO Accession GSM4687563 SRX8811643 GSM4687564 SRP273093 PRJNA647773 SRS7076552 GSM4687564 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687564 GSM4687564 GEO Accession GSM4687564 SRX8811644 GSM4687565 SRP273093 PRJNA647773 SRS7076553 GSM4687565 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687565 GSM4687565 GEO Accession GSM4687565 SRX8811645 GSM4687566 SRP273093 PRJNA647773 SRS7076554 GSM4687566 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687566 GSM4687566 GEO Accession GSM4687566 SRX8811646 GSM4687567 SRP273093 PRJNA647773 SRS7076555 GSM4687567 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687567 GSM4687567 GEO Accession GSM4687567 SRX8811647 GSM4687568 SRP273093 PRJNA647773 SRS7076556 GSM4687568 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687568 GSM4687568 GEO Accession GSM4687568 SRX8811648 GSM4687569 SRP273093 PRJNA647773 SRS7076557 GSM4687569 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687569 GSM4687569 GEO Accession GSM4687569 SRX8811649 GSM4687570 SRP273093 PRJNA647773 SRS7076558 GSM4687570 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687570 GSM4687570 GEO Accession GSM4687570 SRX8811650 GSM4687571 SRP273093 PRJNA647773 SRS7076559 GSM4687571 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687571 GSM4687571 GEO Accession GSM4687571 SRX8811651 GSM4687572 SRP273093 PRJNA647773 SRS7076560 GSM4687572 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687572 GSM4687572 GEO Accession GSM4687572 SRX8811652 GSM4687573 SRP273093 PRJNA647773 SRS7076561 GSM4687573 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687573 GSM4687573 GEO Accession GSM4687573 SRX8811653 GSM4687574 SRP273093 PRJNA647773 SRS7076563 GSM4687574 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687574 GSM4687574 GEO Accession GSM4687574 SRX8811654 GSM4687575 SRP273093 PRJNA647773 SRS7076562 GSM4687575 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687575 GSM4687575 GEO Accession GSM4687575 SRX8811655 GSM4687576 SRP273093 PRJNA647773 SRS7076565 GSM4687576 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687576 GSM4687576 GEO Accession GSM4687576 SRX8811656 GSM4687577 SRP273093 PRJNA647773 SRS7076564 GSM4687577 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687577 GSM4687577 GEO Accession GSM4687577 SRX8811657 GSM4687578 SRP273093 PRJNA647773 SRS7076566 GSM4687578 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687578 GSM4687578 GEO Accession GSM4687578 SRX8811658 GSM4687579 SRP273093 PRJNA647773 SRS7076567 GSM4687579 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687579 GSM4687579 GEO Accession GSM4687579 SRX8811659 GSM4687580 SRP273093 PRJNA647773 SRS7076569 GSM4687580 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687580 GSM4687580 GEO Accession GSM4687580 SRX8811660 GSM4687581 SRP273093 PRJNA647773 SRS7076568 GSM4687581 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687581 GSM4687581 GEO Accession GSM4687581 SRX8811661 GSM4687582 SRP273093 PRJNA647773 SRS7076570 GSM4687582 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687582 GSM4687582 GEO Accession GSM4687582 SRX8811662 GSM4687583 SRP273093 PRJNA647773 SRS7076572 GSM4687583 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687583 GSM4687583 GEO Accession GSM4687583 SRX8811663 GSM4687584 SRP273093 PRJNA647773 SRS7076571 GSM4687584 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687584 GSM4687584 GEO Accession GSM4687584 SRX8811664 GSM4687585 SRP273093 PRJNA647773 SRS7076574 GSM4687585 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687585 GSM4687585 GEO Accession GSM4687585 SRX8811665 GSM4687586 SRP273093 PRJNA647773 SRS7076573 GSM4687586 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687586 GSM4687586 GEO Accession GSM4687586 SRX8811666 GSM4687587 SRP273093 PRJNA647773 SRS7076575 GSM4687587 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687587 GSM4687587 GEO Accession GSM4687587 SRX8811667 GSM4687588 SRP273093 PRJNA647773 SRS7076576 GSM4687588 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687588 GSM4687588 GEO Accession GSM4687588 SRX8811668 GSM4687589 SRP273093 PRJNA647773 SRS7076577 GSM4687589 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687589 GSM4687589 GEO Accession GSM4687589 SRX8811669 GSM4687590 SRP273093 PRJNA647773 SRS7076578 GSM4687590 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687590 GSM4687590 GEO Accession GSM4687590 SRX8811670 GSM4687591 SRP273093 PRJNA647773 SRS7076579 GSM4687591 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687591 GSM4687591 GEO Accession GSM4687591 SRX8811671 GSM4687592 SRP273093 PRJNA647773 SRS7076580 GSM4687592 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687592 GSM4687592 GEO Accession GSM4687592 SRX8811672 GSM4687593 SRP273093 PRJNA647773 SRS7076582 GSM4687593 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687593 GSM4687593 GEO Accession GSM4687593 SRX8811673 GSM4687594 SRP273093 PRJNA647773 SRS7076581 GSM4687594 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687594 GSM4687594 GEO Accession GSM4687594 SRX8811674 GSM4687595 SRP273093 PRJNA647773 SRS7076583 GSM4687595 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687595 GSM4687595 GEO Accession GSM4687595 SRX8811675 GSM4687596 SRP273093 PRJNA647773 SRS7076584 GSM4687596 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687596 GSM4687596 GEO Accession GSM4687596 SRX8811676 GSM4687597 SRP273093 PRJNA647773 SRS7076585 GSM4687597 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687597 GSM4687597 GEO Accession GSM4687597 SRX8811677 GSM4687598 SRP273093 PRJNA647773 SRS7076586 GSM4687598 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687598 GSM4687598 GEO Accession GSM4687598 SRX8811678 GSM4687599 SRP273093 PRJNA647773 SRS7076587 GSM4687599 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687599 GSM4687599 GEO Accession GSM4687599 SRX8811679 GSM4687600 SRP273093 PRJNA647773 SRS7076588 GSM4687600 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687600 GSM4687600 GEO Accession GSM4687600 SRX8811680 GSM4687601 SRP273093 PRJNA647773 SRS7076589 GSM4687601 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687601 GSM4687601 GEO Accession GSM4687601 SRX8811681 GSM4687602 SRP273093 PRJNA647773 SRS7076590 GSM4687602 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687602 GSM4687602 GEO Accession GSM4687602 SRX8811682 GSM4687603 SRP273093 PRJNA647773 SRS7076591 GSM4687603 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687603 GSM4687603 GEO Accession GSM4687603 SRX8811683 GSM4687604 SRP273093 PRJNA647773 SRS7076592 GSM4687604 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687604 GSM4687604 GEO Accession GSM4687604 SRX8811684 GSM4687605 SRP273093 PRJNA647773 SRS7076593 GSM4687605 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687605 GSM4687605 GEO Accession GSM4687605 SRX8811685 GSM4687606 SRP273093 PRJNA647773 SRS7076594 GSM4687606 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687606 GSM4687606 GEO Accession GSM4687606 SRX8811686 GSM4687607 SRP273093 PRJNA647773 SRS7076595 GSM4687607 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687607 GSM4687607 GEO Accession GSM4687607 SRX8811687 GSM4687608 SRP273093 PRJNA647773 SRS7076596 GSM4687608 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687608 GSM4687608 GEO Accession GSM4687608 SRX8811688 GSM4687609 SRP273093 PRJNA647773 SRS7076597 GSM4687609 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687609 GSM4687609 GEO Accession GSM4687609 SRX8811689 GSM4687610 SRP273093 PRJNA647773 SRS7076598 GSM4687610 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687610 GSM4687610 GEO Accession GSM4687610 SRX8811690 GSM4687611 SRP273093 PRJNA647773 SRS7076599 GSM4687611 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687611 GSM4687611 GEO Accession GSM4687611 SRX8811691 GSM4687612 SRP273093 PRJNA647773 SRS7076600 GSM4687612 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687612 GSM4687612 GEO Accession GSM4687612 SRX8811692 GSM4687613 SRP273093 PRJNA647773 SRS7076601 GSM4687613 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687613 GSM4687613 GEO Accession GSM4687613 SRX8811693 GSM4687614 SRP273093 PRJNA647773 SRS7076602 GSM4687614 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687614 GSM4687614 GEO Accession GSM4687614 SRX8811694 GSM4687615 SRP273093 PRJNA647773 SRS7076604 GSM4687615 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687615 GSM4687615 GEO Accession GSM4687615 SRX8811695 GSM4687616 SRP273093 PRJNA647773 SRS7076603 GSM4687616 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687616 GSM4687616 GEO Accession GSM4687616 SRX8811696 GSM4687617 SRP273093 PRJNA647773 SRS7076606 GSM4687617 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687617 GSM4687617 GEO Accession GSM4687617 SRX8811697 GSM4687618 SRP273093 PRJNA647773 SRS7076607 GSM4687618 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687618 GSM4687618 GEO Accession GSM4687618 SRX8811698 GSM4687619 SRP273093 PRJNA647773 SRS7076605 GSM4687619 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687619 GSM4687619 GEO Accession GSM4687619 SRX8811699 GSM4687620 SRP273093 PRJNA647773 SRS7076608 GSM4687620 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687620 GSM4687620 GEO Accession GSM4687620 SRX8811700 GSM4687621 SRP273093 PRJNA647773 SRS7076610 GSM4687621 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687621 GSM4687621 GEO Accession GSM4687621 SRX8811701 GSM4687622 SRP273093 PRJNA647773 SRS7076609 GSM4687622 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687622 GSM4687622 GEO Accession GSM4687622 SRX8811702 GSM4687623 SRP273093 PRJNA647773 SRS7076611 GSM4687623 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687623 GSM4687623 GEO Accession GSM4687623 SRX8811703 GSM4687624 SRP273093 PRJNA647773 SRS7076612 GSM4687624 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687624 GSM4687624 GEO Accession GSM4687624 SRX8811704 GSM4687625 SRP273093 PRJNA647773 SRS7076613 GSM4687625 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687625 GSM4687625 GEO Accession GSM4687625 SRX8811705 GSM4687626 SRP273093 PRJNA647773 SRS7076614 GSM4687626 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687626 GSM4687626 GEO Accession GSM4687626 SRX8811706 GSM4687627 SRP273093 PRJNA647773 SRS7076615 GSM4687627 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687627 GSM4687627 GEO Accession GSM4687627 SRX8811707 GSM4687628 SRP273093 PRJNA647773 SRS7076616 GSM4687628 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687628 GSM4687628 GEO Accession GSM4687628 SRX8811708 GSM4687629 SRP273093 PRJNA647773 SRS7076617 GSM4687629 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687629 GSM4687629 GEO Accession GSM4687629 SRX8811709 GSM4687630 SRP273093 PRJNA647773 SRS7076618 GSM4687630 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687630 GSM4687630 GEO Accession GSM4687630 SRX8811710 GSM4687631 SRP273093 PRJNA647773 SRS7076619 GSM4687631 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687631 GSM4687631 GEO Accession GSM4687631 SRX8811711 GSM4687632 SRP273093 PRJNA647773 SRS7076620 GSM4687632 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687632 GSM4687632 GEO Accession GSM4687632 SRX8811712 GSM4687633 SRP273093 PRJNA647773 SRS7076621 GSM4687633 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687633 GSM4687633 GEO Accession GSM4687633 SRX8811713 GSM4687634 SRP273093 PRJNA647773 SRS7076622 GSM4687634 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687634 GSM4687634 GEO Accession GSM4687634 SRX8811714 GSM4687635 SRP273093 PRJNA647773 SRS7076623 GSM4687635 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687635 GSM4687635 GEO Accession GSM4687635 SRX8811715 GSM4687636 SRP273093 PRJNA647773 SRS7076624 GSM4687636 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687636 GSM4687636 GEO Accession GSM4687636 SRX8811716 GSM4687637 SRP273093 PRJNA647773 SRS7076625 GSM4687637 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687637 GSM4687637 GEO Accession GSM4687637 SRX8811717 GSM4687638 SRP273093 PRJNA647773 SRS7076626 GSM4687638 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687638 GSM4687638 GEO Accession GSM4687638 SRX8811718 GSM4687639 SRP273093 PRJNA647773 SRS7076628 GSM4687639 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687639 GSM4687639 GEO Accession GSM4687639 SRX8811719 GSM4687640 SRP273093 PRJNA647773 SRS7076627 GSM4687640 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687640 GSM4687640 GEO Accession GSM4687640 SRX8811720 GSM4687641 SRP273093 PRJNA647773 SRS7076630 GSM4687641 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687641 GSM4687641 GEO Accession GSM4687641 SRX8811721 GSM4687642 SRP273093 PRJNA647773 SRS7076629 GSM4687642 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687642 GSM4687642 GEO Accession GSM4687642 SRX8811722 GSM4687643 SRP273093 PRJNA647773 SRS7076631 GSM4687643 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687643 GSM4687643 GEO Accession GSM4687643 SRX8811723 GSM4687644 SRP273093 PRJNA647773 SRS7076632 GSM4687644 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687644 GSM4687644 GEO Accession GSM4687644 SRX8811724 GSM4687645 SRP273093 PRJNA647773 SRS7076633 GSM4687645 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687645 GSM4687645 GEO Accession GSM4687645 SRX8811725 GSM4687646 SRP273093 PRJNA647773 SRS7076636 GSM4687646 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687646 GSM4687646 GEO Accession GSM4687646 SRX8811726 GSM4687647 SRP273093 PRJNA647773 SRS7076635 GSM4687647 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687647 GSM4687647 GEO Accession GSM4687647 SRX8811727 GSM4687648 SRP273093 PRJNA647773 SRS7076634 GSM4687648 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687648 GSM4687648 GEO Accession GSM4687648 SRX8811728 GSM4687649 SRP273093 PRJNA647773 SRS7076640 GSM4687649 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687649 GSM4687649 GEO Accession GSM4687649 SRX8811729 GSM4687650 SRP273093 PRJNA647773 SRS7076637 GSM4687650 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687650 GSM4687650 GEO Accession GSM4687650 SRX8811730 GSM4687651 SRP273093 PRJNA647773 SRS7076638 GSM4687651 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687651 GSM4687651 GEO Accession GSM4687651 SRX8811731 GSM4687652 SRP273093 PRJNA647773 SRS7076639 GSM4687652 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687652 GSM4687652 GEO Accession GSM4687652 SRX8811732 GSM4687653 SRP273093 PRJNA647773 SRS7076642 GSM4687653 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687653 GSM4687653 GEO Accession GSM4687653 SRX8811733 GSM4687654 SRP273093 PRJNA647773 SRS7076641 GSM4687654 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687654 GSM4687654 GEO Accession GSM4687654 SRX8811734 GSM4687655 SRP273093 PRJNA647773 SRS7076643 GSM4687655 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687655 GSM4687655 GEO Accession GSM4687655 SRX8811735 GSM4687656 SRP273093 PRJNA647773 SRS7076644 GSM4687656 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687656 GSM4687656 GEO Accession GSM4687656 SRX8811736 GSM4687657 SRP273093 PRJNA647773 SRS7076645 GSM4687657 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687657 GSM4687657 GEO Accession GSM4687657 SRX8811737 GSM4687658 SRP273093 PRJNA647773 SRS7076646 GSM4687658 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687658 GSM4687658 GEO Accession GSM4687658 SRX8811738 GSM4687659 SRP273093 PRJNA647773 SRS7076647 GSM4687659 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687659 GSM4687659 GEO Accession GSM4687659 SRX8811739 GSM4687660 SRP273093 PRJNA647773 SRS7076649 GSM4687660 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687660 GSM4687660 GEO Accession GSM4687660 SRX8811740 GSM4687661 SRP273093 PRJNA647773 SRS7076648 GSM4687661 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687661 GSM4687661 GEO Accession GSM4687661 SRX8811741 GSM4687662 SRP273093 PRJNA647773 SRS7076650 GSM4687662 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687662 GSM4687662 GEO Accession GSM4687662 SRX8811742 GSM4687663 SRP273093 PRJNA647773 SRS7076651 GSM4687663 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687663 GSM4687663 GEO Accession GSM4687663 SRX8811743 GSM4687664 SRP273093 PRJNA647773 SRS7076653 GSM4687664 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687664 GSM4687664 GEO Accession GSM4687664 SRX8811744 GSM4687665 SRP273093 PRJNA647773 SRS7076652 GSM4687665 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687665 GSM4687665 GEO Accession GSM4687665 SRX8811745 GSM4687666 SRP273093 PRJNA647773 SRS7076654 GSM4687666 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687666 GSM4687666 GEO Accession GSM4687666 SRX8811746 GSM4687667 SRP273093 PRJNA647773 SRS7076655 GSM4687667 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687667 GSM4687667 GEO Accession GSM4687667 SRX8811747 GSM4687668 SRP273093 PRJNA647773 SRS7076656 GSM4687668 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687668 GSM4687668 GEO Accession GSM4687668 SRX8811748 GSM4687669 SRP273093 PRJNA647773 SRS7076657 GSM4687669 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687669 GSM4687669 GEO Accession GSM4687669 SRX8811749 GSM4687670 SRP273093 PRJNA647773 SRS7076658 GSM4687670 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687670 GSM4687670 GEO Accession GSM4687670 SRX8811750 GSM4687671 SRP273093 PRJNA647773 SRS7076659 GSM4687671 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687671 GSM4687671 GEO Accession GSM4687671 SRX8811751 GSM4687672 SRP273093 PRJNA647773 SRS7076660 GSM4687672 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687672 GSM4687672 GEO Accession GSM4687672 SRX8811752 GSM4687673 SRP273093 PRJNA647773 SRS7076661 GSM4687673 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687673 GSM4687673 GEO Accession GSM4687673 SRX8811753 GSM4687674 SRP273093 PRJNA647773 SRS7076662 GSM4687674 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687674 GSM4687674 GEO Accession GSM4687674 SRX8811754 GSM4687675 SRP273093 PRJNA647773 SRS7076663 GSM4687675 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687675 GSM4687675 GEO Accession GSM4687675 SRX8811755 GSM4687676 SRP273093 PRJNA647773 SRS7076664 GSM4687676 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687676 GSM4687676 GEO Accession GSM4687676 SRX8811756 GSM4687677 SRP273093 PRJNA647773 SRS7076666 GSM4687677 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687677 GSM4687677 GEO Accession GSM4687677 SRX8811757 GSM4687678 SRP273093 PRJNA647773 SRS7076665 GSM4687678 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687678 GSM4687678 GEO Accession GSM4687678 SRX8811758 GSM4687679 SRP273093 PRJNA647773 SRS7076667 GSM4687679 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687679 GSM4687679 GEO Accession GSM4687679 SRX8811759 GSM4687680 SRP273093 PRJNA647773 SRS7076668 GSM4687680 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687680 GSM4687680 GEO Accession GSM4687680 SRX8811760 GSM4687681 SRP273093 PRJNA647773 SRS7076669 GSM4687681 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687681 GSM4687681 GEO Accession GSM4687681 SRX8811761 GSM4687682 SRP273093 PRJNA647773 SRS7076670 GSM4687682 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687682 GSM4687682 GEO Accession GSM4687682 SRX8811762 GSM4687683 SRP273093 PRJNA647773 SRS7076671 GSM4687683 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687683 GSM4687683 GEO Accession GSM4687683 SRX8811763 GSM4687684 SRP273093 PRJNA647773 SRS7076672 GSM4687684 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687684 GSM4687684 GEO Accession GSM4687684 SRX8811764 GSM4687685 SRP273093 PRJNA647773 SRS7076674 GSM4687685 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687685 GSM4687685 GEO Accession GSM4687685 SRX8811765 GSM4687686 SRP273093 PRJNA647773 SRS7076673 GSM4687686 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687686 GSM4687686 GEO Accession GSM4687686 SRX8811766 GSM4687687 SRP273093 PRJNA647773 SRS7076675 GSM4687687 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687687 GSM4687687 GEO Accession GSM4687687 SRX8811767 GSM4687688 SRP273093 PRJNA647773 SRS7076676 GSM4687688 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687688 GSM4687688 GEO Accession GSM4687688 SRX8811768 GSM4687689 SRP273093 PRJNA647773 SRS7076677 GSM4687689 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687689 GSM4687689 GEO Accession GSM4687689 SRX8811769 GSM4687690 SRP273093 PRJNA647773 SRS7076678 GSM4687690 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687690 GSM4687690 GEO Accession GSM4687690 SRX8811770 GSM4687691 SRP273093 PRJNA647773 SRS7076679 GSM4687691 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687691 GSM4687691 GEO Accession GSM4687691 SRX8811771 GSM4687692 SRP273093 PRJNA647773 SRS7076680 GSM4687692 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687692 GSM4687692 GEO Accession GSM4687692 SRX8811772 GSM4687693 SRP273093 PRJNA647773 SRS7076681 GSM4687693 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687693 GSM4687693 GEO Accession GSM4687693 SRX8811773 GSM4687694 SRP273093 PRJNA647773 SRS7076683 GSM4687694 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687694 GSM4687694 GEO Accession GSM4687694 SRX8811774 GSM4687695 SRP273093 PRJNA647773 SRS7076684 GSM4687695 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687695 GSM4687695 GEO Accession GSM4687695 SRX8811775 GSM4687696 SRP273093 PRJNA647773 SRS7076682 GSM4687696 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687696 GSM4687696 GEO Accession GSM4687696 SRX8811776 GSM4687697 SRP273093 PRJNA647773 SRS7076686 GSM4687697 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687697 GSM4687697 GEO Accession GSM4687697 SRX8811777 GSM4687698 SRP273093 PRJNA647773 SRS7076685 GSM4687698 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687698 GSM4687698 GEO Accession GSM4687698 SRX8811778 GSM4687699 SRP273093 PRJNA647773 SRS7076687 GSM4687699 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687699 GSM4687699 GEO Accession GSM4687699 SRX8811779 GSM4687700 SRP273093 PRJNA647773 SRS7076688 GSM4687700 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687700 GSM4687700 GEO Accession GSM4687700 SRX8811780 GSM4687701 SRP273093 PRJNA647773 SRS7076689 GSM4687701 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687701 GSM4687701 GEO Accession GSM4687701 SRX8811781 GSM4687702 SRP273093 PRJNA647773 SRS7076691 GSM4687702 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687702 GSM4687702 GEO Accession GSM4687702 SRX8811782 GSM4687703 SRP273093 PRJNA647773 SRS7076690 GSM4687703 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687703 GSM4687703 GEO Accession GSM4687703 SRX8811783 GSM4687704 SRP273093 PRJNA647773 SRS7076692 GSM4687704 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687704 GSM4687704 GEO Accession GSM4687704 SRX8811784 GSM4687705 SRP273093 PRJNA647773 SRS7076693 GSM4687705 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687705 GSM4687705 GEO Accession GSM4687705 SRX8811785 GSM4687706 SRP273093 PRJNA647773 SRS7076694 GSM4687706 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687706 GSM4687706 GEO Accession GSM4687706 SRX8811786 GSM4687707 SRP273093 PRJNA647773 SRS7076695 GSM4687707 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687707 GSM4687707 GEO Accession GSM4687707 SRX8811787 GSM4687708 SRP273093 PRJNA647773 SRS7076696 GSM4687708 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687708 GSM4687708 GEO Accession GSM4687708 SRX8811788 GSM4687709 SRP273093 PRJNA647773 SRS7076697 GSM4687709 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687709 GSM4687709 GEO Accession GSM4687709 SRX8811789 GSM4687710 SRP273093 PRJNA647773 SRS7076699 GSM4687710 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687710 GSM4687710 GEO Accession GSM4687710 SRX8811790 GSM4687711 SRP273093 PRJNA647773 SRS7076698 GSM4687711 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687711 GSM4687711 GEO Accession GSM4687711 SRX8811791 GSM4687712 SRP273093 PRJNA647773 SRS7076700 GSM4687712 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687712 GSM4687712 GEO Accession GSM4687712 SRX8811792 GSM4687713 SRP273093 PRJNA647773 SRS7076701 GSM4687713 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687713 GSM4687713 GEO Accession GSM4687713 SRX8811793 GSM4687714 SRP273093 PRJNA647773 SRS7076702 GSM4687714 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687714 GSM4687714 GEO Accession GSM4687714 SRX8811794 GSM4687715 SRP273093 PRJNA647773 SRS7076704 GSM4687715 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687715 GSM4687715 GEO Accession GSM4687715 SRX8811795 GSM4687716 SRP273093 PRJNA647773 SRS7076703 GSM4687716 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687716 GSM4687716 GEO Accession GSM4687716 SRX8811796 GSM4687717 SRP273093 PRJNA647773 SRS7076705 GSM4687717 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687717 GSM4687717 GEO Accession GSM4687717 SRX8811797 GSM4687718 SRP273093 PRJNA647773 SRS7076706 GSM4687718 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687718 GSM4687718 GEO Accession GSM4687718 SRX8811798 GSM4687719 SRP273093 PRJNA647773 SRS7076707 GSM4687719 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687719 GSM4687719 GEO Accession GSM4687719 SRX8811799 GSM4687720 SRP273093 PRJNA647773 SRS7076709 GSM4687720 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687720 GSM4687720 GEO Accession GSM4687720 SRX8811800 GSM4687721 SRP273093 PRJNA647773 SRS7076708 GSM4687721 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687721 GSM4687721 GEO Accession GSM4687721 SRX8811801 GSM4687722 SRP273093 PRJNA647773 SRS7076710 GSM4687722 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687722 GSM4687722 GEO Accession GSM4687722 SRX8811802 GSM4687723 SRP273093 PRJNA647773 SRS7076713 GSM4687723 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687723 GSM4687723 GEO Accession GSM4687723 SRX8811803 GSM4687724 SRP273093 PRJNA647773 SRS7076711 GSM4687724 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687724 GSM4687724 GEO Accession GSM4687724 SRX8811804 GSM4687725 SRP273093 PRJNA647773 SRS7076712 GSM4687725 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687725 GSM4687725 GEO Accession GSM4687725 SRX8811805 GSM4687726 SRP273093 PRJNA647773 SRS7076714 GSM4687726 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687726 GSM4687726 GEO Accession GSM4687726 SRX8811806 GSM4687727 SRP273093 PRJNA647773 SRS7076715 GSM4687727 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687727 GSM4687727 GEO Accession GSM4687727 SRX8811807 GSM4687728 SRP273093 PRJNA647773 SRS7076716 GSM4687728 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687728 GSM4687728 GEO Accession GSM4687728 SRX8811808 GSM4687729 SRP273093 PRJNA647773 SRS7076717 GSM4687729 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687729 GSM4687729 GEO Accession GSM4687729 SRX8811809 GSM4687730 SRP273093 PRJNA647773 SRS7076718 GSM4687730 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687730 GSM4687730 GEO Accession GSM4687730 SRX8811810 GSM4687731 SRP273093 PRJNA647773 SRS7076719 GSM4687731 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687731 GSM4687731 GEO Accession GSM4687731 SRX8811811 GSM4687732 SRP273093 PRJNA647773 SRS7076720 GSM4687732 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687732 GSM4687732 GEO Accession GSM4687732 SRX8811812 GSM4687733 SRP273093 PRJNA647773 SRS7076722 GSM4687733 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687733 GSM4687733 GEO Accession GSM4687733 SRX8811813 GSM4687734 SRP273093 PRJNA647773 SRS7076721 GSM4687734 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687734 GSM4687734 GEO Accession GSM4687734 SRX8811814 GSM4687735 SRP273093 PRJNA647773 SRS7076723 GSM4687735 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687735 GSM4687735 GEO Accession GSM4687735 SRX8811815 GSM4687736 SRP273093 PRJNA647773 SRS7076724 GSM4687736 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687736 GSM4687736 GEO Accession GSM4687736 SRX8811816 GSM4687737 SRP273093 PRJNA647773 SRS7076725 GSM4687737 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687737 GSM4687737 GEO Accession GSM4687737 SRX8811817 GSM4687738 SRP273093 PRJNA647773 SRS7076727 GSM4687738 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687738 GSM4687738 GEO Accession GSM4687738 SRX8811818 GSM4687739 SRP273093 PRJNA647773 SRS7076726 GSM4687739 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687739 GSM4687739 GEO Accession GSM4687739 SRX8811819 GSM4687740 SRP273093 PRJNA647773 SRS7076729 GSM4687740 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687740 GSM4687740 GEO Accession GSM4687740 SRX8811820 GSM4687741 SRP273093 PRJNA647773 SRS7076728 GSM4687741 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687741 GSM4687741 GEO Accession GSM4687741 SRX8811821 GSM4687742 SRP273093 PRJNA647773 SRS7076730 GSM4687742 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687742 GSM4687742 GEO Accession GSM4687742 SRX8811822 GSM4687743 SRP273093 PRJNA647773 SRS7076731 GSM4687743 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687743 GSM4687743 GEO Accession GSM4687743 SRX8811823 GSM4687744 SRP273093 PRJNA647773 SRS7076733 GSM4687744 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687744 GSM4687744 GEO Accession GSM4687744 SRX8811824 GSM4687745 SRP273093 PRJNA647773 SRS7076732 GSM4687745 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687745 GSM4687745 GEO Accession GSM4687745 SRX8811825 GSM4687746 SRP273093 PRJNA647773 SRS7076734 GSM4687746 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687746 GSM4687746 GEO Accession GSM4687746 SRX8811826 GSM4687747 SRP273093 PRJNA647773 SRS7076735 GSM4687747 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687747 GSM4687747 GEO Accession GSM4687747 SRX8811827 GSM4687748 SRP273093 PRJNA647773 SRS7076736 GSM4687748 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687748 GSM4687748 GEO Accession GSM4687748 SRX8811828 GSM4687749 SRP273093 PRJNA647773 SRS7076737 GSM4687749 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687749 GSM4687749 GEO Accession GSM4687749 SRX8811829 GSM4687750 SRP273093 PRJNA647773 SRS7076738 GSM4687750 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687750 GSM4687750 GEO Accession GSM4687750 SRX8811830 GSM4687751 SRP273093 PRJNA647773 SRS7076739 GSM4687751 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687751 GSM4687751 GEO Accession GSM4687751 SRX8811831 GSM4687752 SRP273093 PRJNA647773 SRS7076741 GSM4687752 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687752 GSM4687752 GEO Accession GSM4687752 SRX8811832 GSM4687753 SRP273093 PRJNA647773 SRS7076740 GSM4687753 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687753 GSM4687753 GEO Accession GSM4687753 SRX8811833 GSM4687754 SRP273093 PRJNA647773 SRS7075707 GSM4687754 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687754 GSM4687754 GEO Accession GSM4687754 SRX8811834 GSM4687755 SRP273093 PRJNA647773 SRS7075705 GSM4687755 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687755 GSM4687755 GEO Accession GSM4687755 SRX8811835 GSM4687756 SRP273093 PRJNA647773 SRS7075706 GSM4687756 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687756 GSM4687756 GEO Accession GSM4687756 SRX8811836 GSM4687757 SRP273093 PRJNA647773 SRS7075708 GSM4687757 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687757 GSM4687757 GEO Accession GSM4687757 SRX8811837 GSM4687758 SRP273093 PRJNA647773 SRS7075709 GSM4687758 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687758 GSM4687758 GEO Accession GSM4687758 SRX8811838 GSM4687759 SRP273093 PRJNA647773 SRS7075710 GSM4687759 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687759 GSM4687759 GEO Accession GSM4687759 SRX8811839 GSM4687760 SRP273093 PRJNA647773 SRS7075712 GSM4687760 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687760 GSM4687760 GEO Accession GSM4687760 SRX8811840 GSM4687761 SRP273093 PRJNA647773 SRS7075711 GSM4687761 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687761 GSM4687761 GEO Accession GSM4687761 SRX8811841 GSM4687762 SRP273093 PRJNA647773 SRS7075713 GSM4687762 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687762 GSM4687762 GEO Accession GSM4687762 SRX8811842 GSM4687763 SRP273093 PRJNA647773 SRS7075715 GSM4687763 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687763 GSM4687763 GEO Accession GSM4687763 SRX8811843 GSM4687764 SRP273093 PRJNA647773 SRS7075714 GSM4687764 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687764 GSM4687764 GEO Accession GSM4687764 SRX8811844 GSM4687765 SRP273093 PRJNA647773 SRS7075716 GSM4687765 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687765 GSM4687765 GEO Accession GSM4687765 SRX8811845 GSM4687766 SRP273093 PRJNA647773 SRS7075717 GSM4687766 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687766 GSM4687766 GEO Accession GSM4687766 SRX8811846 GSM4687767 SRP273093 PRJNA647773 SRS7075718 GSM4687767 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687767 GSM4687767 GEO Accession GSM4687767 SRX8811847 GSM4687768 SRP273093 PRJNA647773 SRS7075719 GSM4687768 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687768 GSM4687768 GEO Accession GSM4687768 SRX8811848 GSM4687769 SRP273093 PRJNA647773 SRS7075720 GSM4687769 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687769 GSM4687769 GEO Accession GSM4687769 SRX8811849 GSM4687770 SRP273093 PRJNA647773 SRS7075722 GSM4687770 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687770 GSM4687770 GEO Accession GSM4687770 SRX8811850 GSM4687771 SRP273093 PRJNA647773 SRS7075721 GSM4687771 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687771 GSM4687771 GEO Accession GSM4687771 SRX8811851 GSM4687772 SRP273093 PRJNA647773 SRS7075723 GSM4687772 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687772 GSM4687772 GEO Accession GSM4687772 SRX8811852 GSM4687773 SRP273093 PRJNA647773 SRS7075725 GSM4687773 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687773 GSM4687773 GEO Accession GSM4687773 SRX8811853 GSM4687774 SRP273093 PRJNA647773 SRS7075724 GSM4687774 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687774 GSM4687774 GEO Accession GSM4687774 SRX8811854 GSM4687775 SRP273093 PRJNA647773 SRS7075727 GSM4687775 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687775 GSM4687775 GEO Accession GSM4687775 SRX8811855 GSM4687776 SRP273093 PRJNA647773 SRS7075726 GSM4687776 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687776 GSM4687776 GEO Accession GSM4687776 SRX8811856 GSM4687777 SRP273093 PRJNA647773 SRS7075728 GSM4687777 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687777 GSM4687777 GEO Accession GSM4687777 SRX8811857 GSM4687778 SRP273093 PRJNA647773 SRS7075729 GSM4687778 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687778 GSM4687778 GEO Accession GSM4687778 SRX8811858 GSM4687779 SRP273093 PRJNA647773 SRS7075730 GSM4687779 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687779 GSM4687779 GEO Accession GSM4687779 SRX8811859 GSM4687780 SRP273093 PRJNA647773 SRS7075732 GSM4687780 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687780 GSM4687780 GEO Accession GSM4687780 SRX8811860 GSM4687781 SRP273093 PRJNA647773 SRS7075731 GSM4687781 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687781 GSM4687781 GEO Accession GSM4687781 SRX8811861 GSM4687782 SRP273093 PRJNA647773 SRS7075733 GSM4687782 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687782 GSM4687782 GEO Accession GSM4687782 SRX8811862 GSM4687783 SRP273093 PRJNA647773 SRS7075734 GSM4687783 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687783 GSM4687783 GEO Accession GSM4687783 SRX8811863 GSM4687784 SRP273093 PRJNA647773 SRS7075735 GSM4687784 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687784 GSM4687784 GEO Accession GSM4687784 SRX8811864 GSM4687785 SRP273093 PRJNA647773 SRS7075736 GSM4687785 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687785 GSM4687785 GEO Accession GSM4687785 SRX8811865 GSM4687786 SRP273093 PRJNA647773 SRS7075738 GSM4687786 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687786 GSM4687786 GEO Accession GSM4687786 SRX8811866 GSM4687787 SRP273093 PRJNA647773 SRS7075737 GSM4687787 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687787 GSM4687787 GEO Accession GSM4687787 SRX8811867 GSM4687788 SRP273093 PRJNA647773 SRS7075739 GSM4687788 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687788 GSM4687788 GEO Accession GSM4687788 SRX8811868 GSM4687789 SRP273093 PRJNA647773 SRS7075741 GSM4687789 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687789 GSM4687789 GEO Accession GSM4687789 SRX8811869 GSM4687790 SRP273093 PRJNA647773 SRS7075740 GSM4687790 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687790 GSM4687790 GEO Accession GSM4687790 SRX8811870 GSM4687791 SRP273093 PRJNA647773 SRS7075743 GSM4687791 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687791 GSM4687791 GEO Accession GSM4687791 SRX8811871 GSM4687792 SRP273093 PRJNA647773 SRS7075742 GSM4687792 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687792 GSM4687792 GEO Accession GSM4687792 SRX8811872 GSM4687793 SRP273093 PRJNA647773 SRS7075744 GSM4687793 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687793 GSM4687793 GEO Accession GSM4687793 SRX8811873 GSM4687794 SRP273093 PRJNA647773 SRS7075746 GSM4687794 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687794 GSM4687794 GEO Accession GSM4687794 SRX8811874 GSM4687795 SRP273093 PRJNA647773 SRS7075745 GSM4687795 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687795 GSM4687795 GEO Accession GSM4687795 SRX8811875 GSM4687796 SRP273093 PRJNA647773 SRS7075748 GSM4687796 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687796 GSM4687796 GEO Accession GSM4687796 SRX8811876 GSM4687797 SRP273093 PRJNA647773 SRS7075747 GSM4687797 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687797 GSM4687797 GEO Accession GSM4687797 SRX8811877 GSM4687798 SRP273093 PRJNA647773 SRS7075749 GSM4687798 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687798 GSM4687798 GEO Accession GSM4687798 SRX8811878 GSM4687799 SRP273093 PRJNA647773 SRS7075751 GSM4687799 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687799 GSM4687799 GEO Accession GSM4687799 SRX8811879 GSM4687800 SRP273093 PRJNA647773 SRS7075750 GSM4687800 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687800 GSM4687800 GEO Accession GSM4687800 SRX8811880 GSM4687801 SRP273093 PRJNA647773 SRS7075753 GSM4687801 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687801 GSM4687801 GEO Accession GSM4687801 SRX8811881 GSM4687802 SRP273093 PRJNA647773 SRS7075752 GSM4687802 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687802 GSM4687802 GEO Accession GSM4687802 SRX8811882 GSM4687803 SRP273093 PRJNA647773 SRS7075754 GSM4687803 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687803 GSM4687803 GEO Accession GSM4687803 SRX8811883 GSM4687804 SRP273093 PRJNA647773 SRS7075755 GSM4687804 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687804 GSM4687804 GEO Accession GSM4687804 SRX8811884 GSM4687805 SRP273093 PRJNA647773 SRS7075756 GSM4687805 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687805 GSM4687805 GEO Accession GSM4687805 SRX8811885 GSM4687806 SRP273093 PRJNA647773 SRS7075757 GSM4687806 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687806 GSM4687806 GEO Accession GSM4687806 SRX8811886 GSM4687807 SRP273093 PRJNA647773 SRS7075758 GSM4687807 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687807 GSM4687807 GEO Accession GSM4687807 SRX8811887 GSM4687808 SRP273093 PRJNA647773 SRS7075759 GSM4687808 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687808 GSM4687808 GEO Accession GSM4687808 SRX8811888 GSM4687809 SRP273093 PRJNA647773 SRS7075760 GSM4687809 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687809 GSM4687809 GEO Accession GSM4687809 SRX8811889 GSM4687810 SRP273093 PRJNA647773 SRS7075761 GSM4687810 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687810 GSM4687810 GEO Accession GSM4687810 SRX8811890 GSM4687811 SRP273093 PRJNA647773 SRS7075762 GSM4687811 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687811 GSM4687811 GEO Accession GSM4687811 SRX8811891 GSM4687812 SRP273093 PRJNA647773 SRS7075763 GSM4687812 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687812 GSM4687812 GEO Accession GSM4687812 SRX8811892 GSM4687813 SRP273093 PRJNA647773 SRS7075764 GSM4687813 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687813 GSM4687813 GEO Accession GSM4687813 SRX8811893 GSM4687814 SRP273093 PRJNA647773 SRS7075766 GSM4687814 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687814 GSM4687814 GEO Accession GSM4687814 SRX8811894 GSM4687815 SRP273093 PRJNA647773 SRS7075765 GSM4687815 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687815 GSM4687815 GEO Accession GSM4687815 SRX8811895 GSM4687816 SRP273093 PRJNA647773 SRS7075767 GSM4687816 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687816 GSM4687816 GEO Accession GSM4687816 SRX8811896 GSM4687817 SRP273093 PRJNA647773 SRS7075768 GSM4687817 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687817 GSM4687817 GEO Accession GSM4687817 SRX8811897 GSM4687818 SRP273093 PRJNA647773 SRS7075770 GSM4687818 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687818 GSM4687818 GEO Accession GSM4687818 SRX8811898 GSM4687819 SRP273093 PRJNA647773 SRS7075772 GSM4687819 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687819 GSM4687819 GEO Accession GSM4687819 SRX8811899 GSM4687820 SRP273093 PRJNA647773 SRS7075769 GSM4687820 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687820 GSM4687820 GEO Accession GSM4687820 SRX8811900 GSM4687821 SRP273093 PRJNA647773 SRS7075771 GSM4687821 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687821 GSM4687821 GEO Accession GSM4687821 SRX8811901 GSM4687822 SRP273093 PRJNA647773 SRS7075773 GSM4687822 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687822 GSM4687822 GEO Accession GSM4687822 SRX8811902 GSM4687823 SRP273093 PRJNA647773 SRS7075774 GSM4687823 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687823 GSM4687823 GEO Accession GSM4687823 SRX8811903 GSM4687824 SRP273093 PRJNA647773 SRS7075775 GSM4687824 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687824 GSM4687824 GEO Accession GSM4687824 SRX8811904 GSM4687825 SRP273093 PRJNA647773 SRS7075776 GSM4687825 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687825 GSM4687825 GEO Accession GSM4687825 SRX8811905 GSM4687826 SRP273093 PRJNA647773 SRS7075777 GSM4687826 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687826 GSM4687826 GEO Accession GSM4687826 SRX8811906 GSM4687827 SRP273093 PRJNA647773 SRS7075778 GSM4687827 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687827 GSM4687827 GEO Accession GSM4687827 SRX8811907 GSM4687828 SRP273093 PRJNA647773 SRS7075779 GSM4687828 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687828 GSM4687828 GEO Accession GSM4687828 SRX8811908 GSM4687829 SRP273093 PRJNA647773 SRS7075780 GSM4687829 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687829 GSM4687829 GEO Accession GSM4687829 SRX8811909 GSM4687830 SRP273093 PRJNA647773 SRS7075781 GSM4687830 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687830 GSM4687830 GEO Accession GSM4687830 SRX8811910 GSM4687831 SRP273093 PRJNA647773 SRS7075782 GSM4687831 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687831 GSM4687831 GEO Accession GSM4687831 SRX8811911 GSM4687832 SRP273093 PRJNA647773 SRS7075783 GSM4687832 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687832 GSM4687832 GEO Accession GSM4687832 SRX8811912 GSM4687833 SRP273093 PRJNA647773 SRS7075784 GSM4687833 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687833 GSM4687833 GEO Accession GSM4687833 SRX8811913 GSM4687834 SRP273093 PRJNA647773 SRS7075785 GSM4687834 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687834 GSM4687834 GEO Accession GSM4687834 SRX8811914 GSM4687835 SRP273093 PRJNA647773 SRS7075787 GSM4687835 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687835 GSM4687835 GEO Accession GSM4687835 SRX8811915 GSM4687836 SRP273093 PRJNA647773 SRS7075786 GSM4687836 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687836 GSM4687836 GEO Accession GSM4687836 SRX8811916 GSM4687837 SRP273093 PRJNA647773 SRS7075790 GSM4687837 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687837 GSM4687837 GEO Accession GSM4687837 SRX8811917 GSM4687838 SRP273093 PRJNA647773 SRS7075788 GSM4687838 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687838 GSM4687838 GEO Accession GSM4687838 SRX8811918 GSM4687839 SRP273093 PRJNA647773 SRS7075791 GSM4687839 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687839 GSM4687839 GEO Accession GSM4687839 SRX8811919 GSM4687840 SRP273093 PRJNA647773 SRS7075789 GSM4687840 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687840 GSM4687840 GEO Accession GSM4687840 SRX8811920 GSM4687841 SRP273093 PRJNA647773 SRS7075792 GSM4687841 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687841 GSM4687841 GEO Accession GSM4687841 SRX8811921 GSM4687842 SRP273093 PRJNA647773 SRS7075793 GSM4687842 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687842 GSM4687842 GEO Accession GSM4687842 SRX8811922 GSM4687843 SRP273093 PRJNA647773 SRS7075794 GSM4687843 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687843 GSM4687843 GEO Accession GSM4687843 SRX8811923 GSM4687844 SRP273093 PRJNA647773 SRS7075795 GSM4687844 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687844 GSM4687844 GEO Accession GSM4687844 SRX8811924 GSM4687845 SRP273093 PRJNA647773 SRS7075796 GSM4687845 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687845 GSM4687845 GEO Accession GSM4687845 SRX8811925 GSM4687846 SRP273093 PRJNA647773 SRS7075797 GSM4687846 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687846 GSM4687846 GEO Accession GSM4687846 SRX8811926 GSM4687847 SRP273093 PRJNA647773 SRS7075798 GSM4687847 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687847 GSM4687847 GEO Accession GSM4687847 SRX8811927 GSM4687848 SRP273093 PRJNA647773 SRS7075799 GSM4687848 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687848 GSM4687848 GEO Accession GSM4687848 SRX8811928 GSM4687849 SRP273093 PRJNA647773 SRS7075800 GSM4687849 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687849 GSM4687849 GEO Accession GSM4687849 SRX8811929 GSM4687850 SRP273093 PRJNA647773 SRS7075801 GSM4687850 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687850 GSM4687850 GEO Accession GSM4687850 SRX8811930 GSM4687851 SRP273093 PRJNA647773 SRS7075803 GSM4687851 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687851 GSM4687851 GEO Accession GSM4687851 SRX8811931 GSM4687852 SRP273093 PRJNA647773 SRS7075802 GSM4687852 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687852 GSM4687852 GEO Accession GSM4687852 SRX8811932 GSM4687853 SRP273093 PRJNA647773 SRS7075804 GSM4687853 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687853 GSM4687853 GEO Accession GSM4687853 SRX8811933 GSM4687854 SRP273093 PRJNA647773 SRS7075806 GSM4687854 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687854 GSM4687854 GEO Accession GSM4687854 SRX8811934 GSM4687855 SRP273093 PRJNA647773 SRS7075805 GSM4687855 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687855 GSM4687855 GEO Accession GSM4687855 SRX8811935 GSM4687856 SRP273093 PRJNA647773 SRS7075809 GSM4687856 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687856 GSM4687856 GEO Accession GSM4687856 SRX8811936 GSM4687857 SRP273093 PRJNA647773 SRS7075807 GSM4687857 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687857 GSM4687857 GEO Accession GSM4687857 SRX8811937 GSM4687858 SRP273093 PRJNA647773 SRS7075808 GSM4687858 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687858 GSM4687858 GEO Accession GSM4687858 SRX8811938 GSM4687859 SRP273093 PRJNA647773 SRS7075810 GSM4687859 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687859 GSM4687859 GEO Accession GSM4687859 SRX8811939 GSM4687860 SRP273093 PRJNA647773 SRS7075811 GSM4687860 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687860 GSM4687860 GEO Accession GSM4687860 SRX8811940 GSM4687861 SRP273093 PRJNA647773 SRS7075812 GSM4687861 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687861 GSM4687861 GEO Accession GSM4687861 SRX8811941 GSM4687862 SRP273093 PRJNA647773 SRS7075813 GSM4687862 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687862 GSM4687862 GEO Accession GSM4687862 SRX8811942 GSM4687863 SRP273093 PRJNA647773 SRS7075814 GSM4687863 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687863 GSM4687863 GEO Accession GSM4687863 SRX8811943 GSM4687864 SRP273093 PRJNA647773 SRS7075815 GSM4687864 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687864 GSM4687864 GEO Accession GSM4687864 SRX8811944 GSM4687865 SRP273093 PRJNA647773 SRS7075816 GSM4687865 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687865 GSM4687865 GEO Accession GSM4687865 SRX8811945 GSM4687866 SRP273093 PRJNA647773 SRS7075817 GSM4687866 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687866 GSM4687866 GEO Accession GSM4687866 SRX8811946 GSM4687867 SRP273093 PRJNA647773 SRS7075818 GSM4687867 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687867 GSM4687867 GEO Accession GSM4687867 SRX8811947 GSM4687868 SRP273093 PRJNA647773 SRS7075819 GSM4687868 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687868 GSM4687868 GEO Accession GSM4687868 SRX8811948 GSM4687869 SRP273093 PRJNA647773 SRS7075820 GSM4687869 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687869 GSM4687869 GEO Accession GSM4687869 SRX8811949 GSM4687870 SRP273093 PRJNA647773 SRS7075822 GSM4687870 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687870 GSM4687870 GEO Accession GSM4687870 SRX8811950 GSM4687871 SRP273093 PRJNA647773 SRS7075823 GSM4687871 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687871 GSM4687871 GEO Accession GSM4687871 SRX8811951 GSM4687872 SRP273093 PRJNA647773 SRS7075821 GSM4687872 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687872 GSM4687872 GEO Accession GSM4687872 SRX8811952 GSM4687873 SRP273093 PRJNA647773 SRS7075825 GSM4687873 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687873 GSM4687873 GEO Accession GSM4687873 SRX8811953 GSM4687874 SRP273093 PRJNA647773 SRS7075824 GSM4687874 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687874 GSM4687874 GEO Accession GSM4687874 SRX8811954 GSM4687875 SRP273093 PRJNA647773 SRS7075826 GSM4687875 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687875 GSM4687875 GEO Accession GSM4687875 SRX8811955 GSM4687876 SRP273093 PRJNA647773 SRS7075827 GSM4687876 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687876 GSM4687876 GEO Accession GSM4687876 SRX8811956 GSM4687877 SRP273093 PRJNA647773 SRS7075829 GSM4687877 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687877 GSM4687877 GEO Accession GSM4687877 SRX8811957 GSM4687878 SRP273093 PRJNA647773 SRS7075828 GSM4687878 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687878 GSM4687878 GEO Accession GSM4687878 SRX8811958 GSM4687879 SRP273093 PRJNA647773 SRS7075830 GSM4687879 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687879 GSM4687879 GEO Accession GSM4687879 SRX8811959 GSM4687880 SRP273093 PRJNA647773 SRS7075832 GSM4687880 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687880 GSM4687880 GEO Accession GSM4687880 SRX8811960 GSM4687881 SRP273093 PRJNA647773 SRS7075833 GSM4687881 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687881 GSM4687881 GEO Accession GSM4687881 SRX8811961 GSM4687882 SRP273093 PRJNA647773 SRS7075831 GSM4687882 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687882 GSM4687882 GEO Accession GSM4687882 SRX8811962 GSM4687883 SRP273093 PRJNA647773 SRS7075834 GSM4687883 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687883 GSM4687883 GEO Accession GSM4687883 SRX8811963 GSM4687884 SRP273093 PRJNA647773 SRS7075835 GSM4687884 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687884 GSM4687884 GEO Accession GSM4687884 SRX8811964 GSM4687885 SRP273093 PRJNA647773 SRS7075836 GSM4687885 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687885 GSM4687885 GEO Accession GSM4687885 SRX8811965 GSM4687886 SRP273093 PRJNA647773 SRS7075837 GSM4687886 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687886 GSM4687886 GEO Accession GSM4687886 SRX8811966 GSM4687887 SRP273093 PRJNA647773 SRS7075838 GSM4687887 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687887 GSM4687887 GEO Accession GSM4687887 SRX8811967 GSM4687888 SRP273093 PRJNA647773 SRS7075840 GSM4687888 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687888 GSM4687888 GEO Accession GSM4687888 SRX8811968 GSM4687889 SRP273093 PRJNA647773 SRS7075839 GSM4687889 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687889 GSM4687889 GEO Accession GSM4687889 SRX8811969 GSM4687890 SRP273093 PRJNA647773 SRS7075841 GSM4687890 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687890 GSM4687890 GEO Accession GSM4687890 SRX8811970 GSM4687891 SRP273093 PRJNA647773 SRS7075842 GSM4687891 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687891 GSM4687891 GEO Accession GSM4687891 SRX8811971 GSM4687892 SRP273093 PRJNA647773 SRS7075843 GSM4687892 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687892 GSM4687892 GEO Accession GSM4687892 SRX8811972 GSM4687893 SRP273093 PRJNA647773 SRS7075844 GSM4687893 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687893 GSM4687893 GEO Accession GSM4687893 SRX8811973 GSM4687894 SRP273093 PRJNA647773 SRS7075845 GSM4687894 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687894 GSM4687894 GEO Accession GSM4687894 SRX8811974 GSM4687895 SRP273093 PRJNA647773 SRS7075846 GSM4687895 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687895 GSM4687895 GEO Accession GSM4687895 SRX8811975 GSM4687896 SRP273093 PRJNA647773 SRS7075847 GSM4687896 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687896 GSM4687896 GEO Accession GSM4687896 SRX8811976 GSM4687897 SRP273093 PRJNA647773 SRS7075848 GSM4687897 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687897 GSM4687897 GEO Accession GSM4687897 SRX8811977 GSM4687898 SRP273093 PRJNA647773 SRS7075850 GSM4687898 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687898 GSM4687898 GEO Accession GSM4687898 SRX8811978 GSM4687899 SRP273093 PRJNA647773 SRS7075849 GSM4687899 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687899 GSM4687899 GEO Accession GSM4687899 SRX8811979 GSM4687900 SRP273093 PRJNA647773 SRS7075852 GSM4687900 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687900 GSM4687900 GEO Accession GSM4687900 SRX8811980 GSM4687901 SRP273093 PRJNA647773 SRS7075851 GSM4687901 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687901 GSM4687901 GEO Accession GSM4687901 SRX8811981 GSM4687902 SRP273093 PRJNA647773 SRS7075853 GSM4687902 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687902 GSM4687902 GEO Accession GSM4687902 SRX8811982 GSM4687903 SRP273093 PRJNA647773 SRS7075854 GSM4687903 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687903 GSM4687903 GEO Accession GSM4687903 SRX8811983 GSM4687904 SRP273093 PRJNA647773 SRS7075855 GSM4687904 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687904 GSM4687904 GEO Accession GSM4687904 SRX8811984 GSM4687905 SRP273093 PRJNA647773 SRS7075856 GSM4687905 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687905 GSM4687905 GEO Accession GSM4687905 SRX8811985 GSM4687906 SRP273093 PRJNA647773 SRS7075857 GSM4687906 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687906 GSM4687906 GEO Accession GSM4687906 SRX8811986 GSM4687907 SRP273093 PRJNA647773 SRS7075858 GSM4687907 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687907 GSM4687907 GEO Accession GSM4687907 SRX8811987 GSM4687908 SRP273093 PRJNA647773 SRS7075859 GSM4687908 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687908 GSM4687908 GEO Accession GSM4687908 SRX8811988 GSM4687909 SRP273093 PRJNA647773 SRS7075860 GSM4687909 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687909 GSM4687909 GEO Accession GSM4687909 SRX8811989 GSM4687910 SRP273093 PRJNA647773 SRS7075862 GSM4687910 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687910 GSM4687910 GEO Accession GSM4687910 SRX8811990 GSM4687911 SRP273093 PRJNA647773 SRS7075861 GSM4687911 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687911 GSM4687911 GEO Accession GSM4687911 SRX8811991 GSM4687912 SRP273093 PRJNA647773 SRS7075864 GSM4687912 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687912 GSM4687912 GEO Accession GSM4687912 SRX8811992 GSM4687913 SRP273093 PRJNA647773 SRS7075863 GSM4687913 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687913 GSM4687913 GEO Accession GSM4687913 SRX8811993 GSM4687914 SRP273093 PRJNA647773 SRS7075866 GSM4687914 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687914 GSM4687914 GEO Accession GSM4687914 SRX8811994 GSM4687915 SRP273093 PRJNA647773 SRS7075865 GSM4687915 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687915 GSM4687915 GEO Accession GSM4687915 SRX8811995 GSM4687916 SRP273093 PRJNA647773 SRS7075867 GSM4687916 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687916 GSM4687916 GEO Accession GSM4687916 SRX8811996 GSM4687917 SRP273093 PRJNA647773 SRS7075868 GSM4687917 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687917 GSM4687917 GEO Accession GSM4687917 SRX8811997 GSM4687918 SRP273093 PRJNA647773 SRS7075869 GSM4687918 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687918 GSM4687918 GEO Accession GSM4687918 SRX8811998 GSM4687919 SRP273093 PRJNA647773 SRS7075870 GSM4687919 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687919 GSM4687919 GEO Accession GSM4687919 SRX8811999 GSM4687920 SRP273093 PRJNA647773 SRS7076742 GSM4687920 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687920 GSM4687920 GEO Accession GSM4687920 SRX8812000 GSM4687921 SRP273093 PRJNA647773 SRS7075871 GSM4687921 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687921 GSM4687921 GEO Accession GSM4687921 SRX8812001 GSM4687922 SRP273093 PRJNA647773 SRS7075873 GSM4687922 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687922 GSM4687922 GEO Accession GSM4687922 SRX8812002 GSM4687923 SRP273093 PRJNA647773 SRS7075872 GSM4687923 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687923 GSM4687923 GEO Accession GSM4687923 SRX8812003 GSM4687924 SRP273093 PRJNA647773 SRS7075874 GSM4687924 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687924 GSM4687924 GEO Accession GSM4687924 SRX8812004 GSM4687925 SRP273093 PRJNA647773 SRS7075875 GSM4687925 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687925 GSM4687925 GEO Accession GSM4687925 SRX8812005 GSM4687926 SRP273093 PRJNA647773 SRS7075877 GSM4687926 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687926 GSM4687926 GEO Accession GSM4687926 SRX8812006 GSM4687927 SRP273093 PRJNA647773 SRS7075876 GSM4687927 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687927 GSM4687927 GEO Accession GSM4687927 SRX8812007 GSM4687928 SRP273093 PRJNA647773 SRS7075878 GSM4687928 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687928 GSM4687928 GEO Accession GSM4687928 SRX8812008 GSM4687929 SRP273093 PRJNA647773 SRS7075879 GSM4687929 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687929 GSM4687929 GEO Accession GSM4687929 SRX8812009 GSM4687930 SRP273093 PRJNA647773 SRS7075880 GSM4687930 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687930 GSM4687930 GEO Accession GSM4687930 SRX8812010 GSM4687931 SRP273093 PRJNA647773 SRS7075881 GSM4687931 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687931 GSM4687931 GEO Accession GSM4687931 SRX8812011 GSM4687932 SRP273093 PRJNA647773 SRS7075882 GSM4687932 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687932 GSM4687932 GEO Accession GSM4687932 SRX8812012 GSM4687933 SRP273093 PRJNA647773 SRS7075884 GSM4687933 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687933 GSM4687933 GEO Accession GSM4687933 SRX8812013 GSM4687934 SRP273093 PRJNA647773 SRS7075883 GSM4687934 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687934 GSM4687934 GEO Accession GSM4687934 SRX8812014 GSM4687935 SRP273093 PRJNA647773 SRS7075885 GSM4687935 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687935 GSM4687935 GEO Accession GSM4687935 SRX8812015 GSM4687936 SRP273093 PRJNA647773 SRS7075886 GSM4687936 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687936 GSM4687936 GEO Accession GSM4687936 SRX8812016 GSM4687937 SRP273093 PRJNA647773 SRS7075887 GSM4687937 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687937 GSM4687937 GEO Accession GSM4687937 SRX8812017 GSM4687938 SRP273093 PRJNA647773 SRS7075888 GSM4687938 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687938 GSM4687938 GEO Accession GSM4687938 SRX8812018 GSM4687939 SRP273093 PRJNA647773 SRS7075889 GSM4687939 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687939 GSM4687939 GEO Accession GSM4687939 SRX8812019 GSM4687940 SRP273093 PRJNA647773 SRS7075890 GSM4687940 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687940 GSM4687940 GEO Accession GSM4687940 SRX8812020 GSM4687941 SRP273093 PRJNA647773 SRS7075891 GSM4687941 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687941 GSM4687941 GEO Accession GSM4687941 SRX8812021 GSM4687942 SRP273093 PRJNA647773 SRS7075892 GSM4687942 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687942 GSM4687942 GEO Accession GSM4687942 SRX8812022 GSM4687943 SRP273093 PRJNA647773 SRS7075893 GSM4687943 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687943 GSM4687943 GEO Accession GSM4687943 SRX8812023 GSM4687944 SRP273093 PRJNA647773 SRS7075894 GSM4687944 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687944 GSM4687944 GEO Accession GSM4687944 SRX8812024 GSM4687945 SRP273093 PRJNA647773 SRS7075895 GSM4687945 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687945 GSM4687945 GEO Accession GSM4687945 SRX8812025 GSM4687946 SRP273093 PRJNA647773 SRS7075896 GSM4687946 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687946 GSM4687946 GEO Accession GSM4687946 SRX8812026 GSM4687947 SRP273093 PRJNA647773 SRS7075897 GSM4687947 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687947 GSM4687947 GEO Accession GSM4687947 SRX8812027 GSM4687948 SRP273093 PRJNA647773 SRS7075898 GSM4687948 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687948 GSM4687948 GEO Accession GSM4687948 SRX8812028 GSM4687949 SRP273093 PRJNA647773 SRS7076743 GSM4687949 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687949 GSM4687949 GEO Accession GSM4687949 SRX8812029 GSM4687950 SRP273093 PRJNA647773 SRS7075899 GSM4687950 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687950 GSM4687950 GEO Accession GSM4687950 SRX8812030 GSM4687951 SRP273093 PRJNA647773 SRS7075900 GSM4687951 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687951 GSM4687951 GEO Accession GSM4687951 SRX8812031 GSM4687952 SRP273093 PRJNA647773 SRS7075902 GSM4687952 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687952 GSM4687952 GEO Accession GSM4687952 SRX8812032 GSM4687953 SRP273093 PRJNA647773 SRS7075901 GSM4687953 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687953 GSM4687953 GEO Accession GSM4687953 SRX8812033 GSM4687954 SRP273093 PRJNA647773 SRS7075903 GSM4687954 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687954 GSM4687954 GEO Accession GSM4687954 SRX8812034 GSM4687955 SRP273093 PRJNA647773 SRS7075904 GSM4687955 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687955 GSM4687955 GEO Accession GSM4687955 SRX8812035 GSM4687956 SRP273093 PRJNA647773 SRS7075906 GSM4687956 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687956 GSM4687956 GEO Accession GSM4687956 SRX8812036 GSM4687957 SRP273093 PRJNA647773 SRS7075905 GSM4687957 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687957 GSM4687957 GEO Accession GSM4687957 SRX8812037 GSM4687958 SRP273093 PRJNA647773 SRS7075907 GSM4687958 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687958 GSM4687958 GEO Accession GSM4687958 SRX8812038 GSM4687959 SRP273093 PRJNA647773 SRS7075909 GSM4687959 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687959 GSM4687959 GEO Accession GSM4687959 SRX8812039 GSM4687960 SRP273093 PRJNA647773 SRS7075910 GSM4687960 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687960 GSM4687960 GEO Accession GSM4687960 SRX8812040 GSM4687961 SRP273093 PRJNA647773 SRS7075908 GSM4687961 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687961 GSM4687961 GEO Accession GSM4687961 SRX8812041 GSM4687962 SRP273093 PRJNA647773 SRS7075911 GSM4687962 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687962 GSM4687962 GEO Accession GSM4687962 SRX8812042 GSM4687963 SRP273093 PRJNA647773 SRS7075914 GSM4687963 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687963 GSM4687963 GEO Accession GSM4687963 SRX8812043 GSM4687964 SRP273093 PRJNA647773 SRS7075912 GSM4687964 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687964 GSM4687964 GEO Accession GSM4687964 SRX8812044 GSM4687965 SRP273093 PRJNA647773 SRS7075915 GSM4687965 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687965 GSM4687965 GEO Accession GSM4687965 SRX8812045 GSM4687966 SRP273093 PRJNA647773 SRS7075913 GSM4687966 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687966 GSM4687966 GEO Accession GSM4687966 SRX8812046 GSM4687967 SRP273093 PRJNA647773 SRS7075916 GSM4687967 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687967 GSM4687967 GEO Accession GSM4687967 SRX8812047 GSM4687968 SRP273093 PRJNA647773 SRS7075918 GSM4687968 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687968 GSM4687968 GEO Accession GSM4687968 SRX8812048 GSM4687969 SRP273093 PRJNA647773 SRS7075917 GSM4687969 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687969 GSM4687969 GEO Accession GSM4687969 SRX8812049 GSM4687970 SRP273093 PRJNA647773 SRS7075919 GSM4687970 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687970 GSM4687970 GEO Accession GSM4687970 SRX8812050 GSM4687971 SRP273093 PRJNA647773 SRS7075920 GSM4687971 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687971 GSM4687971 GEO Accession GSM4687971 SRX8812051 GSM4687972 SRP273093 PRJNA647773 SRS7075921 GSM4687972 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687972 GSM4687972 GEO Accession GSM4687972 SRX8812052 GSM4687973 SRP273093 PRJNA647773 SRS7075922 GSM4687973 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687973 GSM4687973 GEO Accession GSM4687973 SRX8812053 GSM4687974 SRP273093 PRJNA647773 SRS7075923 GSM4687974 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687974 GSM4687974 GEO Accession GSM4687974 SRX8812054 GSM4687975 SRP273093 PRJNA647773 SRS7075925 GSM4687975 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687975 GSM4687975 GEO Accession GSM4687975 SRX8812055 GSM4687976 SRP273093 PRJNA647773 SRS7075924 GSM4687976 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687976 GSM4687976 GEO Accession GSM4687976 SRX8812056 GSM4687977 SRP273093 PRJNA647773 SRS7075926 GSM4687977 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687977 GSM4687977 GEO Accession GSM4687977 SRX8812057 GSM4687978 SRP273093 PRJNA647773 SRS7075927 GSM4687978 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687978 GSM4687978 GEO Accession GSM4687978 SRX8812058 GSM4687979 SRP273093 PRJNA647773 SRS7075928 GSM4687979 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687979 GSM4687979 GEO Accession GSM4687979 SRX8812059 GSM4687980 SRP273093 PRJNA647773 SRS7075929 GSM4687980 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687980 GSM4687980 GEO Accession GSM4687980 SRX8812060 GSM4687981 SRP273093 PRJNA647773 SRS7075930 GSM4687981 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687981 GSM4687981 GEO Accession GSM4687981 SRX8812061 GSM4687982 SRP273093 PRJNA647773 SRS7075931 GSM4687982 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687982 GSM4687982 GEO Accession GSM4687982 SRX8812062 GSM4687983 SRP273093 PRJNA647773 SRS7075933 GSM4687983 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687983 GSM4687983 GEO Accession GSM4687983 SRX8812063 GSM4687984 SRP273093 PRJNA647773 SRS7075932 GSM4687984 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687984 GSM4687984 GEO Accession GSM4687984 SRX8812064 GSM4687985 SRP273093 PRJNA647773 SRS7075934 GSM4687985 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687985 GSM4687985 GEO Accession GSM4687985 SRX8812065 GSM4687986 SRP273093 PRJNA647773 SRS7075935 GSM4687986 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687986 GSM4687986 GEO Accession GSM4687986 SRX8812066 GSM4687987 SRP273093 PRJNA647773 SRS7075936 GSM4687987 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687987 GSM4687987 GEO Accession GSM4687987 SRX8812067 GSM4687988 SRP273093 PRJNA647773 SRS7075937 GSM4687988 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687988 GSM4687988 GEO Accession GSM4687988 SRX8812068 GSM4687989 SRP273093 PRJNA647773 SRS7075938 GSM4687989 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687989 GSM4687989 GEO Accession GSM4687989 SRX8812069 GSM4687990 SRP273093 PRJNA647773 SRS7075939 GSM4687990 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687990 GSM4687990 GEO Accession GSM4687990 SRX8812070 GSM4687991 SRP273093 PRJNA647773 SRS7075940 GSM4687991 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687991 GSM4687991 GEO Accession GSM4687991 SRX8812071 GSM4687992 SRP273093 PRJNA647773 SRS7075942 GSM4687992 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687992 GSM4687992 GEO Accession GSM4687992 SRX8812072 GSM4687993 SRP273093 PRJNA647773 SRS7075941 GSM4687993 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687993 GSM4687993 GEO Accession GSM4687993 SRX8812073 GSM4687994 SRP273093 PRJNA647773 SRS7075943 GSM4687994 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687994 GSM4687994 GEO Accession GSM4687994 SRX8812074 GSM4687995 SRP273093 PRJNA647773 SRS7075944 GSM4687995 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687995 GSM4687995 GEO Accession GSM4687995 SRX8812075 GSM4687996 SRP273093 PRJNA647773 SRS7075945 GSM4687996 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687996 GSM4687996 GEO Accession GSM4687996 SRX8812076 GSM4687997 SRP273093 PRJNA647773 SRS7075946 GSM4687997 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687997 GSM4687997 GEO Accession GSM4687997 SRX8812077 GSM4687998 SRP273093 PRJNA647773 SRS7075948 GSM4687998 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687998 GSM4687998 GEO Accession GSM4687998 SRX8812078 GSM4687999 SRP273093 PRJNA647773 SRS7075947 GSM4687999 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304687999 GSM4687999 GEO Accession GSM4687999 SRX8812079 GSM4688000 SRP273093 PRJNA647773 SRS7075949 GSM4688000 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688000 GSM4688000 GEO Accession GSM4688000 SRX8812080 GSM4688001 SRP273093 PRJNA647773 SRS7075950 GSM4688001 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688001 GSM4688001 GEO Accession GSM4688001 SRX8812081 GSM4688002 SRP273093 PRJNA647773 SRS7075951 GSM4688002 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688002 GSM4688002 GEO Accession GSM4688002 SRX8812082 GSM4688003 SRP273093 PRJNA647773 SRS7076744 GSM4688003 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688003 GSM4688003 GEO Accession GSM4688003 SRX8812083 GSM4688004 SRP273093 PRJNA647773 SRS7075952 GSM4688004 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688004 GSM4688004 GEO Accession GSM4688004 SRX8812084 GSM4688005 SRP273093 PRJNA647773 SRS7075953 GSM4688005 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688005 GSM4688005 GEO Accession GSM4688005 SRX8812085 GSM4688006 SRP273093 PRJNA647773 SRS7075954 GSM4688006 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688006 GSM4688006 GEO Accession GSM4688006 SRX8812086 GSM4688007 SRP273093 PRJNA647773 SRS7075955 GSM4688007 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688007 GSM4688007 GEO Accession GSM4688007 SRX8812087 GSM4688008 SRP273093 PRJNA647773 SRS7075956 GSM4688008 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688008 GSM4688008 GEO Accession GSM4688008 SRX8812088 GSM4688009 SRP273093 PRJNA647773 SRS7075957 GSM4688009 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688009 GSM4688009 GEO Accession GSM4688009 SRX8812089 GSM4688010 SRP273093 PRJNA647773 SRS7075958 GSM4688010 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688010 GSM4688010 GEO Accession GSM4688010 SRX8812090 GSM4688011 SRP273093 PRJNA647773 SRS7075959 GSM4688011 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688011 GSM4688011 GEO Accession GSM4688011 SRX8812091 GSM4688012 SRP273093 PRJNA647773 SRS7075960 GSM4688012 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688012 GSM4688012 GEO Accession GSM4688012 SRX8812092 GSM4688013 SRP273093 PRJNA647773 SRS7075961 GSM4688013 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688013 GSM4688013 GEO Accession GSM4688013 SRX8812093 GSM4688014 SRP273093 PRJNA647773 SRS7075962 GSM4688014 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688014 GSM4688014 GEO Accession GSM4688014 SRX8812094 GSM4688015 SRP273093 PRJNA647773 SRS7075963 GSM4688015 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688015 GSM4688015 GEO Accession GSM4688015 SRX8812095 GSM4688016 SRP273093 PRJNA647773 SRS7075964 GSM4688016 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688016 GSM4688016 GEO Accession GSM4688016 SRX8812096 GSM4688017 SRP273093 PRJNA647773 SRS7075965 GSM4688017 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688017 GSM4688017 GEO Accession GSM4688017 SRX8812097 GSM4688018 SRP273093 PRJNA647773 SRS7075966 GSM4688018 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688018 GSM4688018 GEO Accession GSM4688018 SRX8812098 GSM4688019 SRP273093 PRJNA647773 SRS7075967 GSM4688019 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688019 GSM4688019 GEO Accession GSM4688019 SRX8812099 GSM4688020 SRP273093 PRJNA647773 SRS7075968 GSM4688020 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688020 GSM4688020 GEO Accession GSM4688020 SRX8812100 GSM4688021 SRP273093 PRJNA647773 SRS7075969 GSM4688021 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688021 GSM4688021 GEO Accession GSM4688021 SRX8812101 GSM4688022 SRP273093 PRJNA647773 SRS7076745 GSM4688022 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688022 GSM4688022 GEO Accession GSM4688022 SRX8812102 GSM4688023 SRP273093 PRJNA647773 SRS7075970 GSM4688023 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688023 GSM4688023 GEO Accession GSM4688023 SRX8812103 GSM4688024 SRP273093 PRJNA647773 SRS7075971 GSM4688024 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688024 GSM4688024 GEO Accession GSM4688024 SRX8812104 GSM4688025 SRP273093 PRJNA647773 SRS7075972 GSM4688025 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688025 GSM4688025 GEO Accession GSM4688025 SRX8812105 GSM4688026 SRP273093 PRJNA647773 SRS7075973 GSM4688026 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688026 GSM4688026 GEO Accession GSM4688026 SRX8812106 GSM4688027 SRP273093 PRJNA647773 SRS7075974 GSM4688027 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688027 GSM4688027 GEO Accession GSM4688027 SRX8812107 GSM4688028 SRP273093 PRJNA647773 SRS7075975 GSM4688028 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688028 GSM4688028 GEO Accession GSM4688028 SRX8812108 GSM4688029 SRP273093 PRJNA647773 SRS7075976 GSM4688029 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688029 GSM4688029 GEO Accession GSM4688029 SRX8812109 GSM4688030 SRP273093 PRJNA647773 SRS7075978 GSM4688030 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688030 GSM4688030 GEO Accession GSM4688030 SRX8812110 GSM4688031 SRP273093 PRJNA647773 SRS7075977 GSM4688031 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688031 GSM4688031 GEO Accession GSM4688031 SRX8812111 GSM4688032 SRP273093 PRJNA647773 SRS7075979 GSM4688032 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688032 GSM4688032 GEO Accession GSM4688032 SRX8812112 GSM4688033 SRP273093 PRJNA647773 SRS7075980 GSM4688033 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688033 GSM4688033 GEO Accession GSM4688033 SRX8812113 GSM4688034 SRP273093 PRJNA647773 SRS7075981 GSM4688034 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688034 GSM4688034 GEO Accession GSM4688034 SRX8812114 GSM4688035 SRP273093 PRJNA647773 SRS7075982 GSM4688035 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688035 GSM4688035 GEO Accession GSM4688035 SRX8812115 GSM4688036 SRP273093 PRJNA647773 SRS7075984 GSM4688036 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688036 GSM4688036 GEO Accession GSM4688036 SRX8812116 GSM4688037 SRP273093 PRJNA647773 SRS7075983 GSM4688037 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688037 GSM4688037 GEO Accession GSM4688037 SRX8812117 GSM4688038 SRP273093 PRJNA647773 SRS7075985 GSM4688038 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688038 GSM4688038 GEO Accession GSM4688038 SRX8812118 GSM4688039 SRP273093 PRJNA647773 SRS7075986 GSM4688039 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688039 GSM4688039 GEO Accession GSM4688039 SRX8812119 GSM4688040 SRP273093 PRJNA647773 SRS7075988 GSM4688040 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688040 GSM4688040 GEO Accession GSM4688040 SRX8812120 GSM4688041 SRP273093 PRJNA647773 SRS7075987 GSM4688041 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688041 GSM4688041 GEO Accession GSM4688041 SRX8812121 GSM4688042 SRP273093 PRJNA647773 SRS7075989 GSM4688042 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688042 GSM4688042 GEO Accession GSM4688042 SRX8812122 GSM4688043 SRP273093 PRJNA647773 SRS7075990 GSM4688043 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688043 GSM4688043 GEO Accession GSM4688043 SRX8812123 GSM4688044 SRP273093 PRJNA647773 SRS7075991 GSM4688044 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688044 GSM4688044 GEO Accession GSM4688044 SRX8812124 GSM4688045 SRP273093 PRJNA647773 SRS7075992 GSM4688045 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688045 GSM4688045 GEO Accession GSM4688045 SRX8812125 GSM4688046 SRP273093 PRJNA647773 SRS7075993 GSM4688046 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688046 GSM4688046 GEO Accession GSM4688046 SRX8812126 GSM4688047 SRP273093 PRJNA647773 SRS7075994 GSM4688047 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688047 GSM4688047 GEO Accession GSM4688047 SRX8812127 GSM4688048 SRP273093 PRJNA647773 SRS7075995 GSM4688048 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688048 GSM4688048 GEO Accession GSM4688048 SRX8812128 GSM4688049 SRP273093 PRJNA647773 SRS7075996 GSM4688049 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688049 GSM4688049 GEO Accession GSM4688049 SRX8812129 GSM4688050 SRP273093 PRJNA647773 SRS7075999 GSM4688050 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688050 GSM4688050 GEO Accession GSM4688050 SRX8812130 GSM4688051 SRP273093 PRJNA647773 SRS7075997 GSM4688051 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688051 GSM4688051 GEO Accession GSM4688051 SRX8812131 GSM4688052 SRP273093 PRJNA647773 SRS7076000 GSM4688052 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688052 GSM4688052 GEO Accession GSM4688052 SRX8812132 GSM4688053 SRP273093 PRJNA647773 SRS7075998 GSM4688053 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688053 GSM4688053 GEO Accession GSM4688053 SRX8812133 GSM4688054 SRP273093 PRJNA647773 SRS7076001 GSM4688054 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688054 GSM4688054 GEO Accession GSM4688054 SRX8812134 GSM4688055 SRP273093 PRJNA647773 SRS7076002 GSM4688055 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688055 GSM4688055 GEO Accession GSM4688055 SRX8812135 GSM4688056 SRP273093 PRJNA647773 SRS7076003 GSM4688056 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688056 GSM4688056 GEO Accession GSM4688056 SRX8812136 GSM4688057 SRP273093 PRJNA647773 SRS7076005 GSM4688057 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688057 GSM4688057 GEO Accession GSM4688057 SRX8812137 GSM4688058 SRP273093 PRJNA647773 SRS7076004 GSM4688058 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688058 GSM4688058 GEO Accession GSM4688058 SRX8812138 GSM4688059 SRP273093 PRJNA647773 SRS7076006 GSM4688059 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688059 GSM4688059 GEO Accession GSM4688059 SRX8812139 GSM4688060 SRP273093 PRJNA647773 SRS7076007 GSM4688060 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688060 GSM4688060 GEO Accession GSM4688060 SRX8812140 GSM4688061 SRP273093 PRJNA647773 SRS7076008 GSM4688061 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688061 GSM4688061 GEO Accession GSM4688061 SRX8812141 GSM4688062 SRP273093 PRJNA647773 SRS7076009 GSM4688062 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688062 GSM4688062 GEO Accession GSM4688062 SRX8812142 GSM4688063 SRP273093 PRJNA647773 SRS7076010 GSM4688063 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688063 GSM4688063 GEO Accession GSM4688063 SRX8812143 GSM4688064 SRP273093 PRJNA647773 SRS7076011 GSM4688064 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688064 GSM4688064 GEO Accession GSM4688064 SRX8812144 GSM4688065 SRP273093 PRJNA647773 SRS7076013 GSM4688065 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688065 GSM4688065 GEO Accession GSM4688065 SRX8812145 GSM4688066 SRP273093 PRJNA647773 SRS7076012 GSM4688066 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688066 GSM4688066 GEO Accession GSM4688066 SRX8812146 GSM4688067 SRP273093 PRJNA647773 SRS7076014 GSM4688067 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688067 GSM4688067 GEO Accession GSM4688067 SRX8812147 GSM4688068 SRP273093 PRJNA647773 SRS7076015 GSM4688068 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688068 GSM4688068 GEO Accession GSM4688068 SRX8812148 GSM4688069 SRP273093 PRJNA647773 SRS7076016 GSM4688069 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688069 GSM4688069 GEO Accession GSM4688069 SRX8812149 GSM4688070 SRP273093 PRJNA647773 SRS7076017 GSM4688070 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688070 GSM4688070 GEO Accession GSM4688070 SRX8812150 GSM4688071 SRP273093 PRJNA647773 SRS7076018 GSM4688071 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688071 GSM4688071 GEO Accession GSM4688071 SRX8812151 GSM4688072 SRP273093 PRJNA647773 SRS7076019 GSM4688072 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688072 GSM4688072 GEO Accession GSM4688072 SRX8812152 GSM4688073 SRP273093 PRJNA647773 SRS7076020 GSM4688073 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688073 GSM4688073 GEO Accession GSM4688073 SRX8812153 GSM4688074 SRP273093 PRJNA647773 SRS7076021 GSM4688074 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688074 GSM4688074 GEO Accession GSM4688074 SRX8812154 GSM4688075 SRP273093 PRJNA647773 SRS7076022 GSM4688075 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688075 GSM4688075 GEO Accession GSM4688075 SRX8812155 GSM4688076 SRP273093 PRJNA647773 SRS7076024 GSM4688076 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688076 GSM4688076 GEO Accession GSM4688076 SRX8812156 GSM4688077 SRP273093 PRJNA647773 SRS7076023 GSM4688077 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688077 GSM4688077 GEO Accession GSM4688077 SRX8812157 GSM4688078 SRP273093 PRJNA647773 SRS7076026 GSM4688078 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688078 GSM4688078 GEO Accession GSM4688078 SRX8812158 GSM4688079 SRP273093 PRJNA647773 SRS7076025 GSM4688079 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688079 GSM4688079 GEO Accession GSM4688079 SRX8812159 GSM4688080 SRP273093 PRJNA647773 SRS7076027 GSM4688080 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688080 GSM4688080 GEO Accession GSM4688080 SRX8812160 GSM4688081 SRP273093 PRJNA647773 SRS7076028 GSM4688081 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688081 GSM4688081 GEO Accession GSM4688081 SRX8812161 GSM4688082 SRP273093 PRJNA647773 SRS7076029 GSM4688082 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688082 GSM4688082 GEO Accession GSM4688082 SRX8812162 GSM4688083 SRP273093 PRJNA647773 SRS7076031 GSM4688083 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688083 GSM4688083 GEO Accession GSM4688083 SRX8812163 GSM4688084 SRP273093 PRJNA647773 SRS7076030 GSM4688084 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688084 GSM4688084 GEO Accession GSM4688084 SRX8812164 GSM4688085 SRP273093 PRJNA647773 SRS7076033 GSM4688085 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688085 GSM4688085 GEO Accession GSM4688085 SRX8812165 GSM4688086 SRP273093 PRJNA647773 SRS7076032 GSM4688086 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688086 GSM4688086 GEO Accession GSM4688086 SRX8812166 GSM4688087 SRP273093 PRJNA647773 SRS7076034 GSM4688087 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688087 GSM4688087 GEO Accession GSM4688087 SRX8812167 GSM4688088 SRP273093 PRJNA647773 SRS7076035 GSM4688088 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688088 GSM4688088 GEO Accession GSM4688088 SRX8812168 GSM4688089 SRP273093 PRJNA647773 SRS7076036 GSM4688089 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688089 GSM4688089 GEO Accession GSM4688089 SRX8812169 GSM4688090 SRP273093 PRJNA647773 SRS7076038 GSM4688090 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688090 GSM4688090 GEO Accession GSM4688090 SRX8812170 GSM4688091 SRP273093 PRJNA647773 SRS7076037 GSM4688091 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688091 GSM4688091 GEO Accession GSM4688091 SRX8812171 GSM4688092 SRP273093 PRJNA647773 SRS7076039 GSM4688092 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688092 GSM4688092 GEO Accession GSM4688092 SRX8812172 GSM4688093 SRP273093 PRJNA647773 SRS7076040 GSM4688093 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688093 GSM4688093 GEO Accession GSM4688093 SRX8812173 GSM4688094 SRP273093 PRJNA647773 SRS7076041 GSM4688094 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688094 GSM4688094 GEO Accession GSM4688094 SRX8812174 GSM4688095 SRP273093 PRJNA647773 SRS7076042 GSM4688095 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688095 GSM4688095 GEO Accession GSM4688095 SRX8812175 GSM4688096 SRP273093 PRJNA647773 SRS7076044 GSM4688096 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688096 GSM4688096 GEO Accession GSM4688096 SRX8812176 GSM4688097 SRP273093 PRJNA647773 SRS7076043 GSM4688097 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688097 GSM4688097 GEO Accession GSM4688097 SRX8812177 GSM4688098 SRP273093 PRJNA647773 SRS7076045 GSM4688098 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688098 GSM4688098 GEO Accession GSM4688098 SRX8812178 GSM4688099 SRP273093 PRJNA647773 SRS7076046 GSM4688099 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688099 GSM4688099 GEO Accession GSM4688099 SRX8812179 GSM4688100 SRP273093 PRJNA647773 SRS7076047 GSM4688100 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688100 GSM4688100 GEO Accession GSM4688100 SRX8812180 GSM4688101 SRP273093 PRJNA647773 SRS7076049 GSM4688101 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688101 GSM4688101 GEO Accession GSM4688101 SRX8812181 GSM4688102 SRP273093 PRJNA647773 SRS7076048 GSM4688102 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688102 GSM4688102 GEO Accession GSM4688102 SRX8812182 GSM4688103 SRP273093 PRJNA647773 SRS7076050 GSM4688103 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688103 GSM4688103 GEO Accession GSM4688103 SRX8812183 GSM4688104 SRP273093 PRJNA647773 SRS7076051 GSM4688104 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688104 GSM4688104 GEO Accession GSM4688104 SRX8812184 GSM4688105 SRP273093 PRJNA647773 SRS7076052 GSM4688105 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688105 GSM4688105 GEO Accession GSM4688105 SRX8812185 GSM4688106 SRP273093 PRJNA647773 SRS7076053 GSM4688106 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688106 GSM4688106 GEO Accession GSM4688106 SRX8812186 GSM4688107 SRP273093 PRJNA647773 SRS7076054 GSM4688107 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688107 GSM4688107 GEO Accession GSM4688107 SRX8812187 GSM4688108 SRP273093 PRJNA647773 SRS7076056 GSM4688108 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688108 GSM4688108 GEO Accession GSM4688108 SRX8812188 GSM4688109 SRP273093 PRJNA647773 SRS7076055 GSM4688109 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688109 GSM4688109 GEO Accession GSM4688109 SRX8812189 GSM4688110 SRP273093 PRJNA647773 SRS7076057 GSM4688110 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688110 GSM4688110 GEO Accession GSM4688110 SRX8812190 GSM4688111 SRP273093 PRJNA647773 SRS7076058 GSM4688111 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688111 GSM4688111 GEO Accession GSM4688111 SRX8812191 GSM4688112 SRP273093 PRJNA647773 SRS7076060 GSM4688112 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688112 GSM4688112 GEO Accession GSM4688112 SRX8812192 GSM4688113 SRP273093 PRJNA647773 SRS7076059 GSM4688113 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688113 GSM4688113 GEO Accession GSM4688113 SRX8812193 GSM4688114 SRP273093 PRJNA647773 SRS7076061 GSM4688114 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688114 GSM4688114 GEO Accession GSM4688114 SRX8812194 GSM4688115 SRP273093 PRJNA647773 SRS7076062 GSM4688115 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304688115 GSM4688115 GEO Accession GSM4688115 SRX8813518 GSM4689436 SRP273093 PRJNA647773 SRS7077358 GSM4689436 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689436 GSM4689436 GEO Accession GSM4689436 SRX8813519 GSM4689437 SRP273093 PRJNA647773 SRS7077357 GSM4689437 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689437 GSM4689437 GEO Accession GSM4689437 SRX8813520 GSM4689438 SRP273093 PRJNA647773 SRS7077359 GSM4689438 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689438 GSM4689438 GEO Accession GSM4689438 SRX8813521 GSM4689439 SRP273093 PRJNA647773 SRS7077361 GSM4689439 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689439 GSM4689439 GEO Accession GSM4689439 SRX8813522 GSM4689440 SRP273093 PRJNA647773 SRS7077360 GSM4689440 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689440 GSM4689440 GEO Accession GSM4689440 SRX8813523 GSM4689441 SRP273093 PRJNA647773 SRS7077362 GSM4689441 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689441 GSM4689441 GEO Accession GSM4689441 SRX8813524 GSM4689442 SRP273093 PRJNA647773 SRS7077363 GSM4689442 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689442 GSM4689442 GEO Accession GSM4689442 SRX8813525 GSM4689443 SRP273093 PRJNA647773 SRS7077365 GSM4689443 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689443 GSM4689443 GEO Accession GSM4689443 SRX8813526 GSM4689444 SRP273093 PRJNA647773 SRS7077364 GSM4689444 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689444 GSM4689444 GEO Accession GSM4689444 SRX8813527 GSM4689445 SRP273093 PRJNA647773 SRS7077366 GSM4689445 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689445 GSM4689445 GEO Accession GSM4689445 SRX8813528 GSM4689446 SRP273093 PRJNA647773 SRS7077367 GSM4689446 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689446 GSM4689446 GEO Accession GSM4689446 SRX8813529 GSM4689447 SRP273093 PRJNA647773 SRS7077368 GSM4689447 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689447 GSM4689447 GEO Accession GSM4689447 SRX8813530 GSM4689448 SRP273093 PRJNA647773 SRS7077369 GSM4689448 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689448 GSM4689448 GEO Accession GSM4689448 SRX8813531 GSM4689449 SRP273093 PRJNA647773 SRS7077370 GSM4689449 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689449 GSM4689449 GEO Accession GSM4689449 SRX8813532 GSM4689450 SRP273093 PRJNA647773 SRS7077371 GSM4689450 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689450 GSM4689450 GEO Accession GSM4689450 SRX8813533 GSM4689451 SRP273093 PRJNA647773 SRS7077374 GSM4689451 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689451 GSM4689451 GEO Accession GSM4689451 SRX8813534 GSM4689452 SRP273093 PRJNA647773 SRS7077372 GSM4689452 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689452 GSM4689452 GEO Accession GSM4689452 SRX8813535 GSM4689453 SRP273093 PRJNA647773 SRS7077373 GSM4689453 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689453 GSM4689453 GEO Accession GSM4689453 SRX8813536 GSM4689454 SRP273093 PRJNA647773 SRS7077375 GSM4689454 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689454 GSM4689454 GEO Accession GSM4689454 SRX8813537 GSM4689455 SRP273093 PRJNA647773 SRS7077376 GSM4689455 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689455 GSM4689455 GEO Accession GSM4689455 SRX8813538 GSM4689456 SRP273093 PRJNA647773 SRS7077377 GSM4689456 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689456 GSM4689456 GEO Accession GSM4689456 SRX8813539 GSM4689457 SRP273093 PRJNA647773 SRS7077378 GSM4689457 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689457 GSM4689457 GEO Accession GSM4689457 SRX8813540 GSM4689458 SRP273093 PRJNA647773 SRS7077379 GSM4689458 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689458 GSM4689458 GEO Accession GSM4689458 SRX8813541 GSM4689459 SRP273093 PRJNA647773 SRS7077380 GSM4689459 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689459 GSM4689459 GEO Accession GSM4689459 SRX8813542 GSM4689460 SRP273093 PRJNA647773 SRS7077381 GSM4689460 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689460 GSM4689460 GEO Accession GSM4689460 SRX8813543 GSM4689461 SRP273093 PRJNA647773 SRS7077383 GSM4689461 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689461 GSM4689461 GEO Accession GSM4689461 SRX8813544 GSM4689462 SRP273093 PRJNA647773 SRS7077382 GSM4689462 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689462 GSM4689462 GEO Accession GSM4689462 SRX8813545 GSM4689463 SRP273093 PRJNA647773 SRS7077386 GSM4689463 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689463 GSM4689463 GEO Accession GSM4689463 SRX8813546 GSM4689464 SRP273093 PRJNA647773 SRS7077384 GSM4689464 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689464 GSM4689464 GEO Accession GSM4689464 SRX8813547 GSM4689465 SRP273093 PRJNA647773 SRS7077385 GSM4689465 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689465 GSM4689465 GEO Accession GSM4689465 SRX8813548 GSM4689466 SRP273093 PRJNA647773 SRS7077387 GSM4689466 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689466 GSM4689466 GEO Accession GSM4689466 SRX8813549 GSM4689467 SRP273093 PRJNA647773 SRS7077389 GSM4689467 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689467 GSM4689467 GEO Accession GSM4689467 SRX8813550 GSM4689468 SRP273093 PRJNA647773 SRS7077388 GSM4689468 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689468 GSM4689468 GEO Accession GSM4689468 SRX8813551 GSM4689469 SRP273093 PRJNA647773 SRS7077390 GSM4689469 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689469 GSM4689469 GEO Accession GSM4689469 SRX8813552 GSM4689470 SRP273093 PRJNA647773 SRS7077391 GSM4689470 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689470 GSM4689470 GEO Accession GSM4689470 SRX8813553 GSM4689471 SRP273093 PRJNA647773 SRS7077392 GSM4689471 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689471 GSM4689471 GEO Accession GSM4689471 SRX8813554 GSM4689472 SRP273093 PRJNA647773 SRS7077393 GSM4689472 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689472 GSM4689472 GEO Accession GSM4689472 SRX8813555 GSM4689473 SRP273093 PRJNA647773 SRS7077394 GSM4689473 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689473 GSM4689473 GEO Accession GSM4689473 SRX8813556 GSM4689474 SRP273093 PRJNA647773 SRS7077395 GSM4689474 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689474 GSM4689474 GEO Accession GSM4689474 SRX8813557 GSM4689475 SRP273093 PRJNA647773 SRS7077397 GSM4689475 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689475 GSM4689475 GEO Accession GSM4689475 SRX8813558 GSM4689476 SRP273093 PRJNA647773 SRS7077396 GSM4689476 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689476 GSM4689476 GEO Accession GSM4689476 SRX8813559 GSM4689477 SRP273093 PRJNA647773 SRS7077398 GSM4689477 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689477 GSM4689477 GEO Accession GSM4689477 SRX8813560 GSM4689478 SRP273093 PRJNA647773 SRS7077399 GSM4689478 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689478 GSM4689478 GEO Accession GSM4689478 SRX8813561 GSM4689479 SRP273093 PRJNA647773 SRS7077400 GSM4689479 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689479 GSM4689479 GEO Accession GSM4689479 SRX8813562 GSM4689480 SRP273093 PRJNA647773 SRS7077402 GSM4689480 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689480 GSM4689480 GEO Accession GSM4689480 SRX8813563 GSM4689481 SRP273093 PRJNA647773 SRS7077401 GSM4689481 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689481 GSM4689481 GEO Accession GSM4689481 SRX8813564 GSM4689482 SRP273093 PRJNA647773 SRS7077403 GSM4689482 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689482 GSM4689482 GEO Accession GSM4689482 SRX8813565 GSM4689483 SRP273093 PRJNA647773 SRS7077404 GSM4689483 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689483 GSM4689483 GEO Accession GSM4689483 SRX8813566 GSM4689484 SRP273093 PRJNA647773 SRS7077405 GSM4689484 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689484 GSM4689484 GEO Accession GSM4689484 SRX8813567 GSM4689485 SRP273093 PRJNA647773 SRS7077407 GSM4689485 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689485 GSM4689485 GEO Accession GSM4689485 SRX8813568 GSM4689486 SRP273093 PRJNA647773 SRS7077406 GSM4689486 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689486 GSM4689486 GEO Accession GSM4689486 SRX8813569 GSM4689487 SRP273093 PRJNA647773 SRS7077409 GSM4689487 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689487 GSM4689487 GEO Accession GSM4689487 SRX8813570 GSM4689488 SRP273093 PRJNA647773 SRS7077408 GSM4689488 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689488 GSM4689488 GEO Accession GSM4689488 SRX8813571 GSM4689489 SRP273093 PRJNA647773 SRS7077410 GSM4689489 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689489 GSM4689489 GEO Accession GSM4689489 SRX8813572 GSM4689490 SRP273093 PRJNA647773 SRS7077411 GSM4689490 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689490 GSM4689490 GEO Accession GSM4689490 SRX8813573 GSM4689491 SRP273093 PRJNA647773 SRS7077412 GSM4689491 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689491 GSM4689491 GEO Accession GSM4689491 SRX8813574 GSM4689492 SRP273093 PRJNA647773 SRS7077414 GSM4689492 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689492 GSM4689492 GEO Accession GSM4689492 SRX8813575 GSM4689493 SRP273093 PRJNA647773 SRS7077413 GSM4689493 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689493 GSM4689493 GEO Accession GSM4689493 SRX8813576 GSM4689494 SRP273093 PRJNA647773 SRS7077417 GSM4689494 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689494 GSM4689494 GEO Accession GSM4689494 SRX8813577 GSM4689495 SRP273093 PRJNA647773 SRS7077415 GSM4689495 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689495 GSM4689495 GEO Accession GSM4689495 SRX8813578 GSM4689496 SRP273093 PRJNA647773 SRS7077416 GSM4689496 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689496 GSM4689496 GEO Accession GSM4689496 SRX8813579 GSM4689497 SRP273093 PRJNA647773 SRS7077418 GSM4689497 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689497 GSM4689497 GEO Accession GSM4689497 SRX8813580 GSM4689498 SRP273093 PRJNA647773 SRS7077419 GSM4689498 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689498 GSM4689498 GEO Accession GSM4689498 SRX8813581 GSM4689499 SRP273093 PRJNA647773 SRS7077420 GSM4689499 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689499 GSM4689499 GEO Accession GSM4689499 SRX8813582 GSM4689500 SRP273093 PRJNA647773 SRS7077422 GSM4689500 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689500 GSM4689500 GEO Accession GSM4689500 SRX8813583 GSM4689501 SRP273093 PRJNA647773 SRS7077421 GSM4689501 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689501 GSM4689501 GEO Accession GSM4689501 SRX8813584 GSM4689502 SRP273093 PRJNA647773 SRS7077424 GSM4689502 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689502 GSM4689502 GEO Accession GSM4689502 SRX8813585 GSM4689503 SRP273093 PRJNA647773 SRS7077423 GSM4689503 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689503 GSM4689503 GEO Accession GSM4689503 SRX8813586 GSM4689504 SRP273093 PRJNA647773 SRS7077426 GSM4689504 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689504 GSM4689504 GEO Accession GSM4689504 SRX8813587 GSM4689505 SRP273093 PRJNA647773 SRS7077425 GSM4689505 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689505 GSM4689505 GEO Accession GSM4689505 SRX8813588 GSM4689506 SRP273093 PRJNA647773 SRS7077427 GSM4689506 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689506 GSM4689506 GEO Accession GSM4689506 SRX8813589 GSM4689507 SRP273093 PRJNA647773 SRS7077428 GSM4689507 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689507 GSM4689507 GEO Accession GSM4689507 SRX8813590 GSM4689508 SRP273093 PRJNA647773 SRS7077429 GSM4689508 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689508 GSM4689508 GEO Accession GSM4689508 SRX8813591 GSM4689509 SRP273093 PRJNA647773 SRS7077430 GSM4689509 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689509 GSM4689509 GEO Accession GSM4689509 SRX8813592 GSM4689510 SRP273093 PRJNA647773 SRS7077431 GSM4689510 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689510 GSM4689510 GEO Accession GSM4689510 SRX8813593 GSM4689511 SRP273093 PRJNA647773 SRS7077433 GSM4689511 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689511 GSM4689511 GEO Accession GSM4689511 SRX8813594 GSM4689512 SRP273093 PRJNA647773 SRS7077432 GSM4689512 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689512 GSM4689512 GEO Accession GSM4689512 SRX8813595 GSM4689513 SRP273093 PRJNA647773 SRS7077434 GSM4689513 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689513 GSM4689513 GEO Accession GSM4689513 SRX8813596 GSM4689514 SRP273093 PRJNA647773 SRS7077435 GSM4689514 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689514 GSM4689514 GEO Accession GSM4689514 SRX8813597 GSM4689515 SRP273093 PRJNA647773 SRS7077436 GSM4689515 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689515 GSM4689515 GEO Accession GSM4689515 SRX8813598 GSM4689516 SRP273093 PRJNA647773 SRS7077437 GSM4689516 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689516 GSM4689516 GEO Accession GSM4689516 SRX8813599 GSM4689517 SRP273093 PRJNA647773 SRS7077438 GSM4689517 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689517 GSM4689517 GEO Accession GSM4689517 SRX8813600 GSM4689518 SRP273093 PRJNA647773 SRS7077439 GSM4689518 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689518 GSM4689518 GEO Accession GSM4689518 SRX8813601 GSM4689519 SRP273093 PRJNA647773 SRS7077442 GSM4689519 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689519 GSM4689519 GEO Accession GSM4689519 SRX8813602 GSM4689520 SRP273093 PRJNA647773 SRS7077440 GSM4689520 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689520 GSM4689520 GEO Accession GSM4689520 SRX8813603 GSM4689521 SRP273093 PRJNA647773 SRS7077443 GSM4689521 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689521 GSM4689521 GEO Accession GSM4689521 SRX8813604 GSM4689522 SRP273093 PRJNA647773 SRS7077441 GSM4689522 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689522 GSM4689522 GEO Accession GSM4689522 SRX8813605 GSM4689523 SRP273093 PRJNA647773 SRS7077444 GSM4689523 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689523 GSM4689523 GEO Accession GSM4689523 SRX8813606 GSM4689524 SRP273093 PRJNA647773 SRS7077445 GSM4689524 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689524 GSM4689524 GEO Accession GSM4689524 SRX8813607 GSM4689525 SRP273093 PRJNA647773 SRS7077446 GSM4689525 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689525 GSM4689525 GEO Accession GSM4689525 SRX8813608 GSM4689526 SRP273093 PRJNA647773 SRS7077447 GSM4689526 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689526 GSM4689526 GEO Accession GSM4689526 SRX8813609 GSM4689527 SRP273093 PRJNA647773 SRS7077448 GSM4689527 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689527 GSM4689527 GEO Accession GSM4689527 SRX8813610 GSM4689528 SRP273093 PRJNA647773 SRS7077449 GSM4689528 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689528 GSM4689528 GEO Accession GSM4689528 SRX8813611 GSM4689529 SRP273093 PRJNA647773 SRS7077452 GSM4689529 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689529 GSM4689529 GEO Accession GSM4689529 SRX8813612 GSM4689530 SRP273093 PRJNA647773 SRS7077450 GSM4689530 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689530 GSM4689530 GEO Accession GSM4689530 SRX8813613 GSM4689531 SRP273093 PRJNA647773 SRS7077451 GSM4689531 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689531 GSM4689531 GEO Accession GSM4689531 SRX8813614 GSM4689532 SRP273093 PRJNA647773 SRS7077454 GSM4689532 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689532 GSM4689532 GEO Accession GSM4689532 SRX8813615 GSM4689533 SRP273093 PRJNA647773 SRS7077453 GSM4689533 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689533 GSM4689533 GEO Accession GSM4689533 SRX8813616 GSM4689534 SRP273093 PRJNA647773 SRS7077456 GSM4689534 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689534 GSM4689534 GEO Accession GSM4689534 SRX8813617 GSM4689535 SRP273093 PRJNA647773 SRS7077455 GSM4689535 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689535 GSM4689535 GEO Accession GSM4689535 SRX8813618 GSM4689536 SRP273093 PRJNA647773 SRS7077457 GSM4689536 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689536 GSM4689536 GEO Accession GSM4689536 SRX8813619 GSM4689537 SRP273093 PRJNA647773 SRS7077458 GSM4689537 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689537 GSM4689537 GEO Accession GSM4689537 SRX8813620 GSM4689538 SRP273093 PRJNA647773 SRS7077459 GSM4689538 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689538 GSM4689538 GEO Accession GSM4689538 SRX8813621 GSM4689539 SRP273093 PRJNA647773 SRS7077461 GSM4689539 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689539 GSM4689539 GEO Accession GSM4689539 SRX8813622 GSM4689540 SRP273093 PRJNA647773 SRS7077460 GSM4689540 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689540 GSM4689540 GEO Accession GSM4689540 SRX8813623 GSM4689541 SRP273093 PRJNA647773 SRS7077462 GSM4689541 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689541 GSM4689541 GEO Accession GSM4689541 SRX8813624 GSM4689542 SRP273093 PRJNA647773 SRS7077463 GSM4689542 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689542 GSM4689542 GEO Accession GSM4689542 SRX8813625 GSM4689543 SRP273093 PRJNA647773 SRS7077464 GSM4689543 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689543 GSM4689543 GEO Accession GSM4689543 SRX8813626 GSM4689544 SRP273093 PRJNA647773 SRS7077465 GSM4689544 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689544 GSM4689544 GEO Accession GSM4689544 SRX8813627 GSM4689545 SRP273093 PRJNA647773 SRS7077466 GSM4689545 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689545 GSM4689545 GEO Accession GSM4689545 SRX8813628 GSM4689546 SRP273093 PRJNA647773 SRS7077467 GSM4689546 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689546 GSM4689546 GEO Accession GSM4689546 SRX8813629 GSM4689547 SRP273093 PRJNA647773 SRS7077468 GSM4689547 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689547 GSM4689547 GEO Accession GSM4689547 SRX8813630 GSM4689548 SRP273093 PRJNA647773 SRS7077469 GSM4689548 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689548 GSM4689548 GEO Accession GSM4689548 SRX8813631 GSM4689549 SRP273093 PRJNA647773 SRS7077470 GSM4689549 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689549 GSM4689549 GEO Accession GSM4689549 SRX8813632 GSM4689550 SRP273093 PRJNA647773 SRS7077471 GSM4689550 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689550 GSM4689550 GEO Accession GSM4689550 SRX8813633 GSM4689551 SRP273093 PRJNA647773 SRS7077472 GSM4689551 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689551 GSM4689551 GEO Accession GSM4689551 SRX8813634 GSM4689552 SRP273093 PRJNA647773 SRS7077473 GSM4689552 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689552 GSM4689552 GEO Accession GSM4689552 SRX8813635 GSM4689553 SRP273093 PRJNA647773 SRS7077474 GSM4689553 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689553 GSM4689553 GEO Accession GSM4689553 SRX8813636 GSM4689554 SRP273093 PRJNA647773 SRS7077476 GSM4689554 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689554 GSM4689554 GEO Accession GSM4689554 SRX8813637 GSM4689555 SRP273093 PRJNA647773 SRS7077475 GSM4689555 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689555 GSM4689555 GEO Accession GSM4689555 SRX8813638 GSM4689556 SRP273093 PRJNA647773 SRS7077477 GSM4689556 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689556 GSM4689556 GEO Accession GSM4689556 SRX8813639 GSM4689557 SRP273093 PRJNA647773 SRS7077478 GSM4689557 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689557 GSM4689557 GEO Accession GSM4689557 SRX8813641 GSM4689558 SRP273093 PRJNA647773 SRS7077479 GSM4689558 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689558 GSM4689558 GEO Accession GSM4689558 SRX8813642 GSM4689559 SRP273093 PRJNA647773 SRS7077480 GSM4689559 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689559 GSM4689559 GEO Accession GSM4689559 SRX8813643 GSM4689560 SRP273093 PRJNA647773 SRS7077482 GSM4689560 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689560 GSM4689560 GEO Accession GSM4689560 SRX8813644 GSM4689561 SRP273093 PRJNA647773 SRS7077481 GSM4689561 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689561 GSM4689561 GEO Accession GSM4689561 SRX8813645 GSM4689562 SRP273093 PRJNA647773 SRS7077483 GSM4689562 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689562 GSM4689562 GEO Accession GSM4689562 SRX8813646 GSM4689563 SRP273093 PRJNA647773 SRS7077484 GSM4689563 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689563 GSM4689563 GEO Accession GSM4689563 SRX8813647 GSM4689564 SRP273093 PRJNA647773 SRS7077485 GSM4689564 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689564 GSM4689564 GEO Accession GSM4689564 SRX8813648 GSM4689565 SRP273093 PRJNA647773 SRS7077486 GSM4689565 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689565 GSM4689565 GEO Accession GSM4689565 SRX8813649 GSM4689566 SRP273093 PRJNA647773 SRS7077487 GSM4689566 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689566 GSM4689566 GEO Accession GSM4689566 SRX8813650 GSM4689567 SRP273093 PRJNA647773 SRS7077488 GSM4689567 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689567 GSM4689567 GEO Accession GSM4689567 SRX8813651 GSM4689568 SRP273093 PRJNA647773 SRS7077489 GSM4689568 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689568 GSM4689568 GEO Accession GSM4689568 SRX8813652 GSM4689569 SRP273093 PRJNA647773 SRS7077490 GSM4689569 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689569 GSM4689569 GEO Accession GSM4689569 SRX8813653 GSM4689570 SRP273093 PRJNA647773 SRS7077491 GSM4689570 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689570 GSM4689570 GEO Accession GSM4689570 SRX8813654 GSM4689571 SRP273093 PRJNA647773 SRS7077492 GSM4689571 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689571 GSM4689571 GEO Accession GSM4689571 SRX8813655 GSM4689572 SRP273093 PRJNA647773 SRS7077494 GSM4689572 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689572 GSM4689572 GEO Accession GSM4689572 SRX8813656 GSM4689573 SRP273093 PRJNA647773 SRS7077493 GSM4689573 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689573 GSM4689573 GEO Accession GSM4689573 SRX8813657 GSM4689574 SRP273093 PRJNA647773 SRS7077495 GSM4689574 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689574 GSM4689574 GEO Accession GSM4689574 SRX8813658 GSM4689575 SRP273093 PRJNA647773 SRS7077496 GSM4689575 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689575 GSM4689575 GEO Accession GSM4689575 SRX8813659 GSM4689576 SRP273093 PRJNA647773 SRS7077498 GSM4689576 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689576 GSM4689576 GEO Accession GSM4689576 SRX8813660 GSM4689577 SRP273093 PRJNA647773 SRS7077497 GSM4689577 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689577 GSM4689577 GEO Accession GSM4689577 SRX8813661 GSM4689578 SRP273093 PRJNA647773 SRS7077499 GSM4689578 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689578 GSM4689578 GEO Accession GSM4689578 SRX8813662 GSM4689579 SRP273093 PRJNA647773 SRS7077500 GSM4689579 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689579 GSM4689579 GEO Accession GSM4689579 SRX8813663 GSM4689580 SRP273093 PRJNA647773 SRS7077502 GSM4689580 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689580 GSM4689580 GEO Accession GSM4689580 SRX8813664 GSM4689581 SRP273093 PRJNA647773 SRS7077501 GSM4689581 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689581 GSM4689581 GEO Accession GSM4689581 SRX8813665 GSM4689582 SRP273093 PRJNA647773 SRS7077504 GSM4689582 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689582 GSM4689582 GEO Accession GSM4689582 SRX8813666 GSM4689583 SRP273093 PRJNA647773 SRS7077503 GSM4689583 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689583 GSM4689583 GEO Accession GSM4689583 SRX8813667 GSM4689584 SRP273093 PRJNA647773 SRS7077505 GSM4689584 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689584 GSM4689584 GEO Accession GSM4689584 SRX8813668 GSM4689585 SRP273093 PRJNA647773 SRS7077506 GSM4689585 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689585 GSM4689585 GEO Accession GSM4689585 SRX8813669 GSM4689586 SRP273093 PRJNA647773 SRS7077507 GSM4689586 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689586 GSM4689586 GEO Accession GSM4689586 SRX8813670 GSM4689587 SRP273093 PRJNA647773 SRS7077509 GSM4689587 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689587 GSM4689587 GEO Accession GSM4689587 SRX8813671 GSM4689588 SRP273093 PRJNA647773 SRS7077508 GSM4689588 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689588 GSM4689588 GEO Accession GSM4689588 SRX8813673 GSM4689589 SRP273093 PRJNA647773 SRS7077510 GSM4689589 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689589 GSM4689589 GEO Accession GSM4689589 SRX8813674 GSM4689590 SRP273093 PRJNA647773 SRS7077512 GSM4689590 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689590 GSM4689590 GEO Accession GSM4689590 SRX8813675 GSM4689591 SRP273093 PRJNA647773 SRS7077511 GSM4689591 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689591 GSM4689591 GEO Accession GSM4689591 SRX8813676 GSM4689592 SRP273093 PRJNA647773 SRS7077514 GSM4689592 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689592 GSM4689592 GEO Accession GSM4689592 SRX8813677 GSM4689593 SRP273093 PRJNA647773 SRS7077513 GSM4689593 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689593 GSM4689593 GEO Accession GSM4689593 SRX8813678 GSM4689594 SRP273093 PRJNA647773 SRS7077515 GSM4689594 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689594 GSM4689594 GEO Accession GSM4689594 SRX8813679 GSM4689595 SRP273093 PRJNA647773 SRS7077516 GSM4689595 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689595 GSM4689595 GEO Accession GSM4689595 SRX8813680 GSM4689596 SRP273093 PRJNA647773 SRS7077517 GSM4689596 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689596 GSM4689596 GEO Accession GSM4689596 SRX8813681 GSM4689597 SRP273093 PRJNA647773 SRS7077519 GSM4689597 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689597 GSM4689597 GEO Accession GSM4689597 SRX8813682 GSM4689598 SRP273093 PRJNA647773 SRS7077518 GSM4689598 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689598 GSM4689598 GEO Accession GSM4689598 SRX8813683 GSM4689599 SRP273093 PRJNA647773 SRS7077520 GSM4689599 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689599 GSM4689599 GEO Accession GSM4689599 SRX8813684 GSM4689600 SRP273093 PRJNA647773 SRS7077521 GSM4689600 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689600 GSM4689600 GEO Accession GSM4689600 SRX8813685 GSM4689601 SRP273093 PRJNA647773 SRS7077522 GSM4689601 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689601 GSM4689601 GEO Accession GSM4689601 SRX8813686 GSM4689602 SRP273093 PRJNA647773 SRS7077523 GSM4689602 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689602 GSM4689602 GEO Accession GSM4689602 SRX8813687 GSM4689603 SRP273093 PRJNA647773 SRS7077524 GSM4689603 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689603 GSM4689603 GEO Accession GSM4689603 SRX8813688 GSM4689604 SRP273093 PRJNA647773 SRS7077525 GSM4689604 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689604 GSM4689604 GEO Accession GSM4689604 SRX8813689 GSM4689605 SRP273093 PRJNA647773 SRS7077526 GSM4689605 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689605 GSM4689605 GEO Accession GSM4689605 SRX8813690 GSM4689606 SRP273093 PRJNA647773 SRS7077527 GSM4689606 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689606 GSM4689606 GEO Accession GSM4689606 SRX8813691 GSM4689607 SRP273093 PRJNA647773 SRS7077528 GSM4689607 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689607 GSM4689607 GEO Accession GSM4689607 SRX8813692 GSM4689608 SRP273093 PRJNA647773 SRS7077529 GSM4689608 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689608 GSM4689608 GEO Accession GSM4689608 SRX8813693 GSM4689609 SRP273093 PRJNA647773 SRS7077530 GSM4689609 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689609 GSM4689609 GEO Accession GSM4689609 SRX8813694 GSM4689610 SRP273093 PRJNA647773 SRS7077532 GSM4689610 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689610 GSM4689610 GEO Accession GSM4689610 SRX8813695 GSM4689611 SRP273093 PRJNA647773 SRS7077531 GSM4689611 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689611 GSM4689611 GEO Accession GSM4689611 SRX8813696 GSM4689612 SRP273093 PRJNA647773 SRS7077533 GSM4689612 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689612 GSM4689612 GEO Accession GSM4689612 SRX8813697 GSM4689613 SRP273093 PRJNA647773 SRS7077534 GSM4689613 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689613 GSM4689613 GEO Accession GSM4689613 SRX8813698 GSM4689614 SRP273093 PRJNA647773 SRS7077535 GSM4689614 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689614 GSM4689614 GEO Accession GSM4689614 SRX8813699 GSM4689615 SRP273093 PRJNA647773 SRS7077536 GSM4689615 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689615 GSM4689615 GEO Accession GSM4689615 SRX8813700 GSM4689616 SRP273093 PRJNA647773 SRS7077537 GSM4689616 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689616 GSM4689616 GEO Accession GSM4689616 SRX8813701 GSM4689617 SRP273093 PRJNA647773 SRS7077538 GSM4689617 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689617 GSM4689617 GEO Accession GSM4689617 SRX8813702 GSM4689618 SRP273093 PRJNA647773 SRS7077539 GSM4689618 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689618 GSM4689618 GEO Accession GSM4689618 SRX8813703 GSM4689619 SRP273093 PRJNA647773 SRS7077540 GSM4689619 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689619 GSM4689619 GEO Accession GSM4689619 SRX8813704 GSM4689620 SRP273093 PRJNA647773 SRS7077541 GSM4689620 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689620 GSM4689620 GEO Accession GSM4689620 SRX8813705 GSM4689621 SRP273093 PRJNA647773 SRS7077542 GSM4689621 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689621 GSM4689621 GEO Accession GSM4689621 SRX8813706 GSM4689622 SRP273093 PRJNA647773 SRS7077543 GSM4689622 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689622 GSM4689622 GEO Accession GSM4689622 SRX8813707 GSM4689623 SRP273093 PRJNA647773 SRS7077545 GSM4689623 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689623 GSM4689623 GEO Accession GSM4689623 SRX8813708 GSM4689624 SRP273093 PRJNA647773 SRS7077544 GSM4689624 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689624 GSM4689624 GEO Accession GSM4689624 SRX8813709 GSM4689625 SRP273093 PRJNA647773 SRS7077546 GSM4689625 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689625 GSM4689625 GEO Accession GSM4689625 SRX8813710 GSM4689626 SRP273093 PRJNA647773 SRS7077547 GSM4689626 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689626 GSM4689626 GEO Accession GSM4689626 SRX8813711 GSM4689627 SRP273093 PRJNA647773 SRS7077548 GSM4689627 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689627 GSM4689627 GEO Accession GSM4689627 SRX8813712 GSM4689628 SRP273093 PRJNA647773 SRS7077549 GSM4689628 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689628 GSM4689628 GEO Accession GSM4689628 SRX8813713 GSM4689629 SRP273093 PRJNA647773 SRS7077550 GSM4689629 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689629 GSM4689629 GEO Accession GSM4689629 SRX8813714 GSM4689630 SRP273093 PRJNA647773 SRS7077551 GSM4689630 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689630 GSM4689630 GEO Accession GSM4689630 SRX8813715 GSM4689631 SRP273093 PRJNA647773 SRS7077552 GSM4689631 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689631 GSM4689631 GEO Accession GSM4689631 SRX8813716 GSM4689632 SRP273093 PRJNA647773 SRS7077553 GSM4689632 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689632 GSM4689632 GEO Accession GSM4689632 SRX8813717 GSM4689633 SRP273093 PRJNA647773 SRS7077555 GSM4689633 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689633 GSM4689633 GEO Accession GSM4689633 SRX8813718 GSM4689634 SRP273093 PRJNA647773 SRS7077557 GSM4689634 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689634 GSM4689634 GEO Accession GSM4689634 SRX8813719 GSM4689635 SRP273093 PRJNA647773 SRS7077554 GSM4689635 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689635 GSM4689635 GEO Accession GSM4689635 SRX8813720 GSM4689636 SRP273093 PRJNA647773 SRS7077556 GSM4689636 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689636 GSM4689636 GEO Accession GSM4689636 SRX8813721 GSM4689637 SRP273093 PRJNA647773 SRS7077558 GSM4689637 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689637 GSM4689637 GEO Accession GSM4689637 SRX8813722 GSM4689638 SRP273093 PRJNA647773 SRS7077559 GSM4689638 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689638 GSM4689638 GEO Accession GSM4689638 SRX8813723 GSM4689639 SRP273093 PRJNA647773 SRS7077561 GSM4689639 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689639 GSM4689639 GEO Accession GSM4689639 SRX8813724 GSM4689640 SRP273093 PRJNA647773 SRS7077560 GSM4689640 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689640 GSM4689640 GEO Accession GSM4689640 SRX8813725 GSM4689641 SRP273093 PRJNA647773 SRS7077564 GSM4689641 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689641 GSM4689641 GEO Accession GSM4689641 SRX8813726 GSM4689642 SRP273093 PRJNA647773 SRS7077562 GSM4689642 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689642 GSM4689642 GEO Accession GSM4689642 SRX8813727 GSM4689643 SRP273093 PRJNA647773 SRS7077563 GSM4689643 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689643 GSM4689643 GEO Accession GSM4689643 SRX8813728 GSM4689644 SRP273093 PRJNA647773 SRS7077565 GSM4689644 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689644 GSM4689644 GEO Accession GSM4689644 SRX8813729 GSM4689645 SRP273093 PRJNA647773 SRS7077566 GSM4689645 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689645 GSM4689645 GEO Accession GSM4689645 SRX8813730 GSM4689646 SRP273093 PRJNA647773 SRS7077567 GSM4689646 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689646 GSM4689646 GEO Accession GSM4689646 SRX8813731 GSM4689647 SRP273093 PRJNA647773 SRS7077568 GSM4689647 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689647 GSM4689647 GEO Accession GSM4689647 SRX8813732 GSM4689648 SRP273093 PRJNA647773 SRS7077569 GSM4689648 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689648 GSM4689648 GEO Accession GSM4689648 SRX8813733 GSM4689649 SRP273093 PRJNA647773 SRS7077570 GSM4689649 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689649 GSM4689649 GEO Accession GSM4689649 SRX8813734 GSM4689650 SRP273093 PRJNA647773 SRS7077571 GSM4689650 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689650 GSM4689650 GEO Accession GSM4689650 SRX8813735 GSM4689651 SRP273093 PRJNA647773 SRS7077572 GSM4689651 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689651 GSM4689651 GEO Accession GSM4689651 SRX8813736 GSM4689652 SRP273093 PRJNA647773 SRS7077574 GSM4689652 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689652 GSM4689652 GEO Accession GSM4689652 SRX8813737 GSM4689653 SRP273093 PRJNA647773 SRS7077575 GSM4689653 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689653 GSM4689653 GEO Accession GSM4689653 SRX8813738 GSM4689654 SRP273093 PRJNA647773 SRS7077573 GSM4689654 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689654 GSM4689654 GEO Accession GSM4689654 SRX8813739 GSM4689655 SRP273093 PRJNA647773 SRS7077576 GSM4689655 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689655 GSM4689655 GEO Accession GSM4689655 SRX8813740 GSM4689656 SRP273093 PRJNA647773 SRS7077577 GSM4689656 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689656 GSM4689656 GEO Accession GSM4689656 SRX8813741 GSM4689657 SRP273093 PRJNA647773 SRS7077578 GSM4689657 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689657 GSM4689657 GEO Accession GSM4689657 SRX8813742 GSM4689658 SRP273093 PRJNA647773 SRS7077579 GSM4689658 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689658 GSM4689658 GEO Accession GSM4689658 SRX8813743 GSM4689659 SRP273093 PRJNA647773 SRS7077582 GSM4689659 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689659 GSM4689659 GEO Accession GSM4689659 SRX8813744 GSM4689660 SRP273093 PRJNA647773 SRS7077580 GSM4689660 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689660 GSM4689660 GEO Accession GSM4689660 SRX8813745 GSM4689661 SRP273093 PRJNA647773 SRS7077581 GSM4689661 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689661 GSM4689661 GEO Accession GSM4689661 SRX8813746 GSM4689662 SRP273093 PRJNA647773 SRS7077583 GSM4689662 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689662 GSM4689662 GEO Accession GSM4689662 SRX8813747 GSM4689663 SRP273093 PRJNA647773 SRS7077584 GSM4689663 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689663 GSM4689663 GEO Accession GSM4689663 SRX8813748 GSM4689664 SRP273093 PRJNA647773 SRS7077586 GSM4689664 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689664 GSM4689664 GEO Accession GSM4689664 SRX8813749 GSM4689665 SRP273093 PRJNA647773 SRS7077585 GSM4689665 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689665 GSM4689665 GEO Accession GSM4689665 SRX8813750 GSM4689666 SRP273093 PRJNA647773 SRS7077587 GSM4689666 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689666 GSM4689666 GEO Accession GSM4689666 SRX8813751 GSM4689667 SRP273093 PRJNA647773 SRS7077588 GSM4689667 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689667 GSM4689667 GEO Accession GSM4689667 SRX8813752 GSM4689668 SRP273093 PRJNA647773 SRS7077589 GSM4689668 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689668 GSM4689668 GEO Accession GSM4689668 SRX8813753 GSM4689669 SRP273093 PRJNA647773 SRS7077590 GSM4689669 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689669 GSM4689669 GEO Accession GSM4689669 SRX8813754 GSM4689670 SRP273093 PRJNA647773 SRS7077591 GSM4689670 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689670 GSM4689670 GEO Accession GSM4689670 SRX8813755 GSM4689671 SRP273093 PRJNA647773 SRS7077592 GSM4689671 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689671 GSM4689671 GEO Accession GSM4689671 SRX8813756 GSM4689672 SRP273093 PRJNA647773 SRS7077593 GSM4689672 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689672 GSM4689672 GEO Accession GSM4689672 SRX8813757 GSM4689673 SRP273093 PRJNA647773 SRS7077594 GSM4689673 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689673 GSM4689673 GEO Accession GSM4689673 SRX8813758 GSM4689674 SRP273093 PRJNA647773 SRS7077595 GSM4689674 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689674 GSM4689674 GEO Accession GSM4689674 SRX8813759 GSM4689675 SRP273093 PRJNA647773 SRS7077596 GSM4689675 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689675 GSM4689675 GEO Accession GSM4689675 SRX8813760 GSM4689676 SRP273093 PRJNA647773 SRS7077597 GSM4689676 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689676 GSM4689676 GEO Accession GSM4689676 SRX8813761 GSM4689677 SRP273093 PRJNA647773 SRS7077598 GSM4689677 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689677 GSM4689677 GEO Accession GSM4689677 SRX8813762 GSM4689678 SRP273093 PRJNA647773 SRS7077599 GSM4689678 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689678 GSM4689678 GEO Accession GSM4689678 SRX8813763 GSM4689679 SRP273093 PRJNA647773 SRS7077600 GSM4689679 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689679 GSM4689679 GEO Accession GSM4689679 SRX8813764 GSM4689680 SRP273093 PRJNA647773 SRS7077601 GSM4689680 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689680 GSM4689680 GEO Accession GSM4689680 SRX8813765 GSM4689681 SRP273093 PRJNA647773 SRS7077602 GSM4689681 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689681 GSM4689681 GEO Accession GSM4689681 SRX8813766 GSM4689682 SRP273093 PRJNA647773 SRS7077603 GSM4689682 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689682 GSM4689682 GEO Accession GSM4689682 SRX8813767 GSM4689683 SRP273093 PRJNA647773 SRS7077604 GSM4689683 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689683 GSM4689683 GEO Accession GSM4689683 SRX8813768 GSM4689684 SRP273093 PRJNA647773 SRS7077605 GSM4689684 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689684 GSM4689684 GEO Accession GSM4689684 SRX8813769 GSM4689685 SRP273093 PRJNA647773 SRS7077606 GSM4689685 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689685 GSM4689685 GEO Accession GSM4689685 SRX8813770 GSM4689686 SRP273093 PRJNA647773 SRS7077608 GSM4689686 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689686 GSM4689686 GEO Accession GSM4689686 SRX8813771 GSM4689687 SRP273093 PRJNA647773 SRS7077607 GSM4689687 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689687 GSM4689687 GEO Accession GSM4689687 SRX8813772 GSM4689688 SRP273093 PRJNA647773 SRS7077609 GSM4689688 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689688 GSM4689688 GEO Accession GSM4689688 SRX8813773 GSM4689689 SRP273093 PRJNA647773 SRS7077611 GSM4689689 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689689 GSM4689689 GEO Accession GSM4689689 SRX8813774 GSM4689690 SRP273093 PRJNA647773 SRS7077610 GSM4689690 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689690 GSM4689690 GEO Accession GSM4689690 SRX8813775 GSM4689691 SRP273093 PRJNA647773 SRS7077612 GSM4689691 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689691 GSM4689691 GEO Accession GSM4689691 SRX8813776 GSM4689692 SRP273093 PRJNA647773 SRS7077613 GSM4689692 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689692 GSM4689692 GEO Accession GSM4689692 SRX8813777 GSM4689693 SRP273093 PRJNA647773 SRS7077614 GSM4689693 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689693 GSM4689693 GEO Accession GSM4689693 SRX8813778 GSM4689694 SRP273093 PRJNA647773 SRS7077615 GSM4689694 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689694 GSM4689694 GEO Accession GSM4689694 SRX8813779 GSM4689695 SRP273093 PRJNA647773 SRS7077616 GSM4689695 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689695 GSM4689695 GEO Accession GSM4689695 SRX8813780 GSM4689696 SRP273093 PRJNA647773 SRS7077617 GSM4689696 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689696 GSM4689696 GEO Accession GSM4689696 SRX8813781 GSM4689697 SRP273093 PRJNA647773 SRS7077618 GSM4689697 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689697 GSM4689697 GEO Accession GSM4689697 SRX8813782 GSM4689698 SRP273093 PRJNA647773 SRS7077619 GSM4689698 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689698 GSM4689698 GEO Accession GSM4689698 SRX8813783 GSM4689699 SRP273093 PRJNA647773 SRS7077620 GSM4689699 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689699 GSM4689699 GEO Accession GSM4689699 SRX8813784 GSM4689700 SRP273093 PRJNA647773 SRS7077621 GSM4689700 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689700 GSM4689700 GEO Accession GSM4689700 SRX8813785 GSM4689701 SRP273093 PRJNA647773 SRS7077622 GSM4689701 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689701 GSM4689701 GEO Accession GSM4689701 SRX8813786 GSM4689702 SRP273093 PRJNA647773 SRS7077623 GSM4689702 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689702 GSM4689702 GEO Accession GSM4689702 SRX8813787 GSM4689703 SRP273093 PRJNA647773 SRS7077624 GSM4689703 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689703 GSM4689703 GEO Accession GSM4689703 SRX8813788 GSM4689704 SRP273093 PRJNA647773 SRS7077625 GSM4689704 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689704 GSM4689704 GEO Accession GSM4689704 SRX8813789 GSM4689705 SRP273093 PRJNA647773 SRS7077626 GSM4689705 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689705 GSM4689705 GEO Accession GSM4689705 SRX8813790 GSM4689706 SRP273093 PRJNA647773 SRS7077628 GSM4689706 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689706 GSM4689706 GEO Accession GSM4689706 SRX8813791 GSM4689707 SRP273093 PRJNA647773 SRS7077627 GSM4689707 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689707 GSM4689707 GEO Accession GSM4689707 SRX8813792 GSM4689708 SRP273093 PRJNA647773 SRS7077629 GSM4689708 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689708 GSM4689708 GEO Accession GSM4689708 SRX8813793 GSM4689709 SRP273093 PRJNA647773 SRS7077630 GSM4689709 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689709 GSM4689709 GEO Accession GSM4689709 SRX8813794 GSM4689710 SRP273093 PRJNA647773 SRS7077631 GSM4689710 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689710 GSM4689710 GEO Accession GSM4689710 SRX8813795 GSM4689711 SRP273093 PRJNA647773 SRS7077632 GSM4689711 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689711 GSM4689711 GEO Accession GSM4689711 SRX8813796 GSM4689712 SRP273093 PRJNA647773 SRS7077633 GSM4689712 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689712 GSM4689712 GEO Accession GSM4689712 SRX8813797 GSM4689713 SRP273093 PRJNA647773 SRS7077634 GSM4689713 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689713 GSM4689713 GEO Accession GSM4689713 SRX8813798 GSM4689714 SRP273093 PRJNA647773 SRS7077635 GSM4689714 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689714 GSM4689714 GEO Accession GSM4689714 SRX8813799 GSM4689715 SRP273093 PRJNA647773 SRS7077638 GSM4689715 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689715 GSM4689715 GEO Accession GSM4689715 SRX8813800 GSM4689716 SRP273093 PRJNA647773 SRS7077636 GSM4689716 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689716 GSM4689716 GEO Accession GSM4689716 SRX8813801 GSM4689717 SRP273093 PRJNA647773 SRS7077637 GSM4689717 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689717 GSM4689717 GEO Accession GSM4689717 SRX8813802 GSM4689718 SRP273093 PRJNA647773 SRS7077639 GSM4689718 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689718 GSM4689718 GEO Accession GSM4689718 SRX8813803 GSM4689719 SRP273093 PRJNA647773 SRS7077640 GSM4689719 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689719 GSM4689719 GEO Accession GSM4689719 SRX8813804 GSM4689720 SRP273093 PRJNA647773 SRS7077641 GSM4689720 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689720 GSM4689720 GEO Accession GSM4689720 SRX8813805 GSM4689721 SRP273093 PRJNA647773 SRS7077642 GSM4689721 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689721 GSM4689721 GEO Accession GSM4689721 SRX8813806 GSM4689722 SRP273093 PRJNA647773 SRS7077643 GSM4689722 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689722 GSM4689722 GEO Accession GSM4689722 SRX8813807 GSM4689723 SRP273093 PRJNA647773 SRS7077644 GSM4689723 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689723 GSM4689723 GEO Accession GSM4689723 SRX8813808 GSM4689724 SRP273093 PRJNA647773 SRS7077645 GSM4689724 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689724 GSM4689724 GEO Accession GSM4689724 SRX8813809 GSM4689725 SRP273093 PRJNA647773 SRS7077646 GSM4689725 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689725 GSM4689725 GEO Accession GSM4689725 SRX8813810 GSM4689726 SRP273093 PRJNA647773 SRS7077647 GSM4689726 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689726 GSM4689726 GEO Accession GSM4689726 SRX8813811 GSM4689727 SRP273093 PRJNA647773 SRS7077648 GSM4689727 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689727 GSM4689727 GEO Accession GSM4689727 SRX8813812 GSM4689728 SRP273093 PRJNA647773 SRS7077649 GSM4689728 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689728 GSM4689728 GEO Accession GSM4689728 SRX8813813 GSM4689729 SRP273093 PRJNA647773 SRS7076746 GSM4689729 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689729 GSM4689729 GEO Accession GSM4689729 SRX8813814 GSM4689730 SRP273093 PRJNA647773 SRS7077650 GSM4689730 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689730 GSM4689730 GEO Accession GSM4689730 SRX8813815 GSM4689731 SRP273093 PRJNA647773 SRS7077651 GSM4689731 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689731 GSM4689731 GEO Accession GSM4689731 SRX8813816 GSM4689732 SRP273093 PRJNA647773 SRS7077652 GSM4689732 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689732 GSM4689732 GEO Accession GSM4689732 SRX8813817 GSM4689733 SRP273093 PRJNA647773 SRS7077654 GSM4689733 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689733 GSM4689733 GEO Accession GSM4689733 SRX8813818 GSM4689734 SRP273093 PRJNA647773 SRS7077653 GSM4689734 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689734 GSM4689734 GEO Accession GSM4689734 SRX8813819 GSM4689735 SRP273093 PRJNA647773 SRS7077655 GSM4689735 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689735 GSM4689735 GEO Accession GSM4689735 SRX8813820 GSM4689736 SRP273093 PRJNA647773 SRS7077656 GSM4689736 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689736 GSM4689736 GEO Accession GSM4689736 SRX8813821 GSM4689737 SRP273093 PRJNA647773 SRS7077657 GSM4689737 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689737 GSM4689737 GEO Accession GSM4689737 SRX8813822 GSM4689738 SRP273093 PRJNA647773 SRS7077658 GSM4689738 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689738 GSM4689738 GEO Accession GSM4689738 SRX8813823 GSM4689739 SRP273093 PRJNA647773 SRS7077659 GSM4689739 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689739 GSM4689739 GEO Accession GSM4689739 SRX8813824 GSM4689740 SRP273093 PRJNA647773 SRS7077660 GSM4689740 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689740 GSM4689740 GEO Accession GSM4689740 SRX8813825 GSM4689741 SRP273093 PRJNA647773 SRS7077661 GSM4689741 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689741 GSM4689741 GEO Accession GSM4689741 SRX8813826 GSM4689742 SRP273093 PRJNA647773 SRS7077662 GSM4689742 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689742 GSM4689742 GEO Accession GSM4689742 SRX8813827 GSM4689743 SRP273093 PRJNA647773 SRS7077663 GSM4689743 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689743 GSM4689743 GEO Accession GSM4689743 SRX8813828 GSM4689744 SRP273093 PRJNA647773 SRS7077664 GSM4689744 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689744 GSM4689744 GEO Accession GSM4689744 SRX8813829 GSM4689745 SRP273093 PRJNA647773 SRS7077665 GSM4689745 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689745 GSM4689745 GEO Accession GSM4689745 SRX8813830 GSM4689746 SRP273093 PRJNA647773 SRS7077666 GSM4689746 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689746 GSM4689746 GEO Accession GSM4689746 SRX8813831 GSM4689747 SRP273093 PRJNA647773 SRS7077667 GSM4689747 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689747 GSM4689747 GEO Accession GSM4689747 SRX8813832 GSM4689748 SRP273093 PRJNA647773 SRS7077668 GSM4689748 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689748 GSM4689748 GEO Accession GSM4689748 SRX8813833 GSM4689749 SRP273093 PRJNA647773 SRS7077670 GSM4689749 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689749 GSM4689749 GEO Accession GSM4689749 SRX8813834 GSM4689750 SRP273093 PRJNA647773 SRS7077669 GSM4689750 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689750 GSM4689750 GEO Accession GSM4689750 SRX8813835 GSM4689751 SRP273093 PRJNA647773 SRS7077671 GSM4689751 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689751 GSM4689751 GEO Accession GSM4689751 SRX8813836 GSM4689752 SRP273093 PRJNA647773 SRS7077672 GSM4689752 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689752 GSM4689752 GEO Accession GSM4689752 SRX8813837 GSM4689753 SRP273093 PRJNA647773 SRS7077673 GSM4689753 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689753 GSM4689753 GEO Accession GSM4689753 SRX8813838 GSM4689754 SRP273093 PRJNA647773 SRS7077674 GSM4689754 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689754 GSM4689754 GEO Accession GSM4689754 SRX8813839 GSM4689755 SRP273093 PRJNA647773 SRS7077675 GSM4689755 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689755 GSM4689755 GEO Accession GSM4689755 SRX8813840 GSM4689756 SRP273093 PRJNA647773 SRS7077676 GSM4689756 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689756 GSM4689756 GEO Accession GSM4689756 SRX8813841 GSM4689757 SRP273093 PRJNA647773 SRS7077677 GSM4689757 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689757 GSM4689757 GEO Accession GSM4689757 SRX8813842 GSM4689758 SRP273093 PRJNA647773 SRS7077678 GSM4689758 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689758 GSM4689758 GEO Accession GSM4689758 SRX8813843 GSM4689759 SRP273093 PRJNA647773 SRS7077680 GSM4689759 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689759 GSM4689759 GEO Accession GSM4689759 SRX8813844 GSM4689760 SRP273093 PRJNA647773 SRS7077679 GSM4689760 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689760 GSM4689760 GEO Accession GSM4689760 SRX8813845 GSM4689761 SRP273093 PRJNA647773 SRS7077681 GSM4689761 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689761 GSM4689761 GEO Accession GSM4689761 SRX8813846 GSM4689762 SRP273093 PRJNA647773 SRS7077682 GSM4689762 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689762 GSM4689762 GEO Accession GSM4689762 SRX8813847 GSM4689763 SRP273093 PRJNA647773 SRS7077683 GSM4689763 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689763 GSM4689763 GEO Accession GSM4689763 SRX8813848 GSM4689764 SRP273093 PRJNA647773 SRS7077684 GSM4689764 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689764 GSM4689764 GEO Accession GSM4689764 SRX8813849 GSM4689765 SRP273093 PRJNA647773 SRS7077685 GSM4689765 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689765 GSM4689765 GEO Accession GSM4689765 SRX8813850 GSM4689766 SRP273093 PRJNA647773 SRS7077686 GSM4689766 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689766 GSM4689766 GEO Accession GSM4689766 SRX8813851 GSM4689767 SRP273093 PRJNA647773 SRS7077687 GSM4689767 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689767 GSM4689767 GEO Accession GSM4689767 SRX8813852 GSM4689768 SRP273093 PRJNA647773 SRS7077688 GSM4689768 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689768 GSM4689768 GEO Accession GSM4689768 SRX8813853 GSM4689769 SRP273093 PRJNA647773 SRS7077689 GSM4689769 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689769 GSM4689769 GEO Accession GSM4689769 SRX8813854 GSM4689770 SRP273093 PRJNA647773 SRS7077690 GSM4689770 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689770 GSM4689770 GEO Accession GSM4689770 SRX8813855 GSM4689771 SRP273093 PRJNA647773 SRS7077691 GSM4689771 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689771 GSM4689771 GEO Accession GSM4689771 SRX8813856 GSM4689772 SRP273093 PRJNA647773 SRS7077692 GSM4689772 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689772 GSM4689772 GEO Accession GSM4689772 SRX8813857 GSM4689773 SRP273093 PRJNA647773 SRS7077693 GSM4689773 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689773 GSM4689773 GEO Accession GSM4689773 SRX8813858 GSM4689774 SRP273093 PRJNA647773 SRS7077694 GSM4689774 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689774 GSM4689774 GEO Accession GSM4689774 SRX8813859 GSM4689775 SRP273093 PRJNA647773 SRS7077695 GSM4689775 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689775 GSM4689775 GEO Accession GSM4689775 SRX8813860 GSM4689776 SRP273093 PRJNA647773 SRS7077696 GSM4689776 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689776 GSM4689776 GEO Accession GSM4689776 SRX8813861 GSM4689777 SRP273093 PRJNA647773 SRS7077697 GSM4689777 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689777 GSM4689777 GEO Accession GSM4689777 SRX8813862 GSM4689778 SRP273093 PRJNA647773 SRS7077698 GSM4689778 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689778 GSM4689778 GEO Accession GSM4689778 SRX8813863 GSM4689779 SRP273093 PRJNA647773 SRS7077699 GSM4689779 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689779 GSM4689779 GEO Accession GSM4689779 SRX8813864 GSM4689780 SRP273093 PRJNA647773 SRS7077700 GSM4689780 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689780 GSM4689780 GEO Accession GSM4689780 SRX8813865 GSM4689781 SRP273093 PRJNA647773 SRS7077701 GSM4689781 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689781 GSM4689781 GEO Accession GSM4689781 SRX8813866 GSM4689782 SRP273093 PRJNA647773 SRS7077703 GSM4689782 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689782 GSM4689782 GEO Accession GSM4689782 SRX8813867 GSM4689783 SRP273093 PRJNA647773 SRS7077702 GSM4689783 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689783 GSM4689783 GEO Accession GSM4689783 SRX8813868 GSM4689784 SRP273093 PRJNA647773 SRS7077705 GSM4689784 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689784 GSM4689784 GEO Accession GSM4689784 SRX8813869 GSM4689785 SRP273093 PRJNA647773 SRS7077704 GSM4689785 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689785 GSM4689785 GEO Accession GSM4689785 SRX8813870 GSM4689786 SRP273093 PRJNA647773 SRS7077706 GSM4689786 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689786 GSM4689786 GEO Accession GSM4689786 SRX8813871 GSM4689787 SRP273093 PRJNA647773 SRS7077707 GSM4689787 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689787 GSM4689787 GEO Accession GSM4689787 SRX8813872 GSM4689788 SRP273093 PRJNA647773 SRS7077709 GSM4689788 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689788 GSM4689788 GEO Accession GSM4689788 SRX8813873 GSM4689789 SRP273093 PRJNA647773 SRS7077708 GSM4689789 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689789 GSM4689789 GEO Accession GSM4689789 SRX8813874 GSM4689790 SRP273093 PRJNA647773 SRS7077710 GSM4689790 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689790 GSM4689790 GEO Accession GSM4689790 SRX8813875 GSM4689791 SRP273093 PRJNA647773 SRS7077711 GSM4689791 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689791 GSM4689791 GEO Accession GSM4689791 SRX8813876 GSM4689792 SRP273093 PRJNA647773 SRS7077713 GSM4689792 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689792 GSM4689792 GEO Accession GSM4689792 SRX8813877 GSM4689793 SRP273093 PRJNA647773 SRS7077712 GSM4689793 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689793 GSM4689793 GEO Accession GSM4689793 SRX8813878 GSM4689794 SRP273093 PRJNA647773 SRS7077715 GSM4689794 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689794 GSM4689794 GEO Accession GSM4689794 SRX8813879 GSM4689795 SRP273093 PRJNA647773 SRS7077714 GSM4689795 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689795 GSM4689795 GEO Accession GSM4689795 SRX8813880 GSM4689796 SRP273093 PRJNA647773 SRS7077716 GSM4689796 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689796 GSM4689796 GEO Accession GSM4689796 SRX8813881 GSM4689797 SRP273093 PRJNA647773 SRS7077717 GSM4689797 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689797 GSM4689797 GEO Accession GSM4689797 SRX8813882 GSM4689798 SRP273093 PRJNA647773 SRS7077718 GSM4689798 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689798 GSM4689798 GEO Accession GSM4689798 SRX8813883 GSM4689799 SRP273093 PRJNA647773 SRS7077719 GSM4689799 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689799 GSM4689799 GEO Accession GSM4689799 SRX8813884 GSM4689800 SRP273093 PRJNA647773 SRS7077720 GSM4689800 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689800 GSM4689800 GEO Accession GSM4689800 SRX8813885 GSM4689801 SRP273093 PRJNA647773 SRS7077721 GSM4689801 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689801 GSM4689801 GEO Accession GSM4689801 SRX8813886 GSM4689802 SRP273093 PRJNA647773 SRS7077722 GSM4689802 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689802 GSM4689802 GEO Accession GSM4689802 SRX8813887 GSM4689803 SRP273093 PRJNA647773 SRS7077723 GSM4689803 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689803 GSM4689803 GEO Accession GSM4689803 SRX8813888 GSM4689804 SRP273093 PRJNA647773 SRS7077725 GSM4689804 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689804 GSM4689804 GEO Accession GSM4689804 SRX8813889 GSM4689805 SRP273093 PRJNA647773 SRS7077724 GSM4689805 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689805 GSM4689805 GEO Accession GSM4689805 SRX8813890 GSM4689806 SRP273093 PRJNA647773 SRS7077726 GSM4689806 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689806 GSM4689806 GEO Accession GSM4689806 SRX8813891 GSM4689807 SRP273093 PRJNA647773 SRS7077727 GSM4689807 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689807 GSM4689807 GEO Accession GSM4689807 SRX8813892 GSM4689808 SRP273093 PRJNA647773 SRS7077729 GSM4689808 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689808 GSM4689808 GEO Accession GSM4689808 SRX8813893 GSM4689809 SRP273093 PRJNA647773 SRS7077728 GSM4689809 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689809 GSM4689809 GEO Accession GSM4689809 SRX8813894 GSM4689810 SRP273093 PRJNA647773 SRS7077731 GSM4689810 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689810 GSM4689810 GEO Accession GSM4689810 SRX8813895 GSM4689811 SRP273093 PRJNA647773 SRS7077730 GSM4689811 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689811 GSM4689811 GEO Accession GSM4689811 SRX8813896 GSM4689812 SRP273093 PRJNA647773 SRS7077732 GSM4689812 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689812 GSM4689812 GEO Accession GSM4689812 SRX8813897 GSM4689813 SRP273093 PRJNA647773 SRS7077733 GSM4689813 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689813 GSM4689813 GEO Accession GSM4689813 SRX8813898 GSM4689814 SRP273093 PRJNA647773 SRS7077734 GSM4689814 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689814 GSM4689814 GEO Accession GSM4689814 SRX8813899 GSM4689815 SRP273093 PRJNA647773 SRS7077736 GSM4689815 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689815 GSM4689815 GEO Accession GSM4689815 SRX8813900 GSM4689816 SRP273093 PRJNA647773 SRS7077735 GSM4689816 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689816 GSM4689816 GEO Accession GSM4689816 SRX8813901 GSM4689817 SRP273093 PRJNA647773 SRS7077739 GSM4689817 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689817 GSM4689817 GEO Accession GSM4689817 SRX8813902 GSM4689818 SRP273093 PRJNA647773 SRS7077738 GSM4689818 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689818 GSM4689818 GEO Accession GSM4689818 SRX8813903 GSM4689819 SRP273093 PRJNA647773 SRS7077737 GSM4689819 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689819 GSM4689819 GEO Accession GSM4689819 SRX8813904 GSM4689820 SRP273093 PRJNA647773 SRS7077740 GSM4689820 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689820 GSM4689820 GEO Accession GSM4689820 SRX8813905 GSM4689821 SRP273093 PRJNA647773 SRS7077741 GSM4689821 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689821 GSM4689821 GEO Accession GSM4689821 SRX8813906 GSM4689822 SRP273093 PRJNA647773 SRS7077742 GSM4689822 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689822 GSM4689822 GEO Accession GSM4689822 SRX8813907 GSM4689823 SRP273093 PRJNA647773 SRS7077744 GSM4689823 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689823 GSM4689823 GEO Accession GSM4689823 SRX8813908 GSM4689824 SRP273093 PRJNA647773 SRS7077743 GSM4689824 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689824 GSM4689824 GEO Accession GSM4689824 SRX8813909 GSM4689825 SRP273093 PRJNA647773 SRS7077746 GSM4689825 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689825 GSM4689825 GEO Accession GSM4689825 SRX8813910 GSM4689826 SRP273093 PRJNA647773 SRS7077745 GSM4689826 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689826 GSM4689826 GEO Accession GSM4689826 SRX8813911 GSM4689827 SRP273093 PRJNA647773 SRS7077747 GSM4689827 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689827 GSM4689827 GEO Accession GSM4689827 SRX8813912 GSM4689828 SRP273093 PRJNA647773 SRS7077748 GSM4689828 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689828 GSM4689828 GEO Accession GSM4689828 SRX8813913 GSM4689829 SRP273093 PRJNA647773 SRS7077749 GSM4689829 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689829 GSM4689829 GEO Accession GSM4689829 SRX8813914 GSM4689830 SRP273093 PRJNA647773 SRS7077751 GSM4689830 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689830 GSM4689830 GEO Accession GSM4689830 SRX8813915 GSM4689831 SRP273093 PRJNA647773 SRS7077750 GSM4689831 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689831 GSM4689831 GEO Accession GSM4689831 SRX8813916 GSM4689832 SRP273093 PRJNA647773 SRS7077752 GSM4689832 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689832 GSM4689832 GEO Accession GSM4689832 SRX8813917 GSM4689833 SRP273093 PRJNA647773 SRS7077754 GSM4689833 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689833 GSM4689833 GEO Accession GSM4689833 SRX8813918 GSM4689834 SRP273093 PRJNA647773 SRS7077756 GSM4689834 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689834 GSM4689834 GEO Accession GSM4689834 SRX8813919 GSM4689835 SRP273093 PRJNA647773 SRS7077753 GSM4689835 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689835 GSM4689835 GEO Accession GSM4689835 SRX8813920 GSM4689836 SRP273093 PRJNA647773 SRS7077755 GSM4689836 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689836 GSM4689836 GEO Accession GSM4689836 SRX8813921 GSM4689837 SRP273093 PRJNA647773 SRS7077757 GSM4689837 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689837 GSM4689837 GEO Accession GSM4689837 SRX8813922 GSM4689838 SRP273093 PRJNA647773 SRS7077758 GSM4689838 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689838 GSM4689838 GEO Accession GSM4689838 SRX8813923 GSM4689839 SRP273093 PRJNA647773 SRS7077762 GSM4689839 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689839 GSM4689839 GEO Accession GSM4689839 SRX8813924 GSM4689840 SRP273093 PRJNA647773 SRS7077760 GSM4689840 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689840 GSM4689840 GEO Accession GSM4689840 SRX8813925 GSM4689841 SRP273093 PRJNA647773 SRS7077759 GSM4689841 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689841 GSM4689841 GEO Accession GSM4689841 SRX8813926 GSM4689842 SRP273093 PRJNA647773 SRS7077764 GSM4689842 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689842 GSM4689842 GEO Accession GSM4689842 SRX8813927 GSM4689843 SRP273093 PRJNA647773 SRS7077767 GSM4689843 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689843 GSM4689843 GEO Accession GSM4689843 SRX8813928 GSM4689844 SRP273093 PRJNA647773 SRS7077763 GSM4689844 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689844 GSM4689844 GEO Accession GSM4689844 SRX8813929 GSM4689845 SRP273093 PRJNA647773 SRS7077766 GSM4689845 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689845 GSM4689845 GEO Accession GSM4689845 SRX8813930 GSM4689846 SRP273093 PRJNA647773 SRS7077761 GSM4689846 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689846 GSM4689846 GEO Accession GSM4689846 SRX8813931 GSM4689847 SRP273093 PRJNA647773 SRS7077765 GSM4689847 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689847 GSM4689847 GEO Accession GSM4689847 SRX8813932 GSM4689848 SRP273093 PRJNA647773 SRS7077770 GSM4689848 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689848 GSM4689848 GEO Accession GSM4689848 SRX8813933 GSM4689849 SRP273093 PRJNA647773 SRS7077768 GSM4689849 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689849 GSM4689849 GEO Accession GSM4689849 SRX8813934 GSM4689850 SRP273093 PRJNA647773 SRS7077769 GSM4689850 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689850 GSM4689850 GEO Accession GSM4689850 SRX8813935 GSM4689851 SRP273093 PRJNA647773 SRS7077771 GSM4689851 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689851 GSM4689851 GEO Accession GSM4689851 SRX8813936 GSM4689852 SRP273093 PRJNA647773 SRS7077772 GSM4689852 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689852 GSM4689852 GEO Accession GSM4689852 SRX8813937 GSM4689853 SRP273093 PRJNA647773 SRS7077773 GSM4689853 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689853 GSM4689853 GEO Accession GSM4689853 SRX8813938 GSM4689854 SRP273093 PRJNA647773 SRS7077776 GSM4689854 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689854 GSM4689854 GEO Accession GSM4689854 SRX8813939 GSM4689855 SRP273093 PRJNA647773 SRS7077774 GSM4689855 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689855 GSM4689855 GEO Accession GSM4689855 SRX8813940 GSM4689856 SRP273093 PRJNA647773 SRS7077775 GSM4689856 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689856 GSM4689856 GEO Accession GSM4689856 SRX8813941 GSM4689857 SRP273093 PRJNA647773 SRS7077777 GSM4689857 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689857 GSM4689857 GEO Accession GSM4689857 SRX8813942 GSM4689858 SRP273093 PRJNA647773 SRS7077778 GSM4689858 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689858 GSM4689858 GEO Accession GSM4689858 SRX8813943 GSM4689859 SRP273093 PRJNA647773 SRS7077779 GSM4689859 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689859 GSM4689859 GEO Accession GSM4689859 SRX8813944 GSM4689860 SRP273093 PRJNA647773 SRS7077780 GSM4689860 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689860 GSM4689860 GEO Accession GSM4689860 SRX8813945 GSM4689861 SRP273093 PRJNA647773 SRS7077784 GSM4689861 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689861 GSM4689861 GEO Accession GSM4689861 SRX8813946 GSM4689862 SRP273093 PRJNA647773 SRS7077781 GSM4689862 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689862 GSM4689862 GEO Accession GSM4689862 SRX8813947 GSM4689863 SRP273093 PRJNA647773 SRS7077783 GSM4689863 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689863 GSM4689863 GEO Accession GSM4689863 SRX8813948 GSM4689864 SRP273093 PRJNA647773 SRS7077782 GSM4689864 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689864 GSM4689864 GEO Accession GSM4689864 SRX8813949 GSM4689865 SRP273093 PRJNA647773 SRS7077785 GSM4689865 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689865 GSM4689865 GEO Accession GSM4689865 SRX8813950 GSM4689866 SRP273093 PRJNA647773 SRS7077787 GSM4689866 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689866 GSM4689866 GEO Accession GSM4689866 SRX8813951 GSM4689867 SRP273093 PRJNA647773 SRS7077786 GSM4689867 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689867 GSM4689867 GEO Accession GSM4689867 SRX8813952 GSM4689868 SRP273093 PRJNA647773 SRS7077788 GSM4689868 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689868 GSM4689868 GEO Accession GSM4689868 SRX8813953 GSM4689869 SRP273093 PRJNA647773 SRS7077789 GSM4689869 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689869 GSM4689869 GEO Accession GSM4689869 SRX8813954 GSM4689870 SRP273093 PRJNA647773 SRS7077791 GSM4689870 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689870 GSM4689870 GEO Accession GSM4689870 SRX8813955 GSM4689871 SRP273093 PRJNA647773 SRS7077790 GSM4689871 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689871 GSM4689871 GEO Accession GSM4689871 SRX8813956 GSM4689872 SRP273093 PRJNA647773 SRS7077792 GSM4689872 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689872 GSM4689872 GEO Accession GSM4689872 SRX8813957 GSM4689873 SRP273093 PRJNA647773 SRS7077793 GSM4689873 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689873 GSM4689873 GEO Accession GSM4689873 SRX8813958 GSM4689874 SRP273093 PRJNA647773 SRS7077794 GSM4689874 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689874 GSM4689874 GEO Accession GSM4689874 SRX8813959 GSM4689875 SRP273093 PRJNA647773 SRS7077795 GSM4689875 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689875 GSM4689875 GEO Accession GSM4689875 SRX8813960 GSM4689876 SRP273093 PRJNA647773 SRS7077796 GSM4689876 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689876 GSM4689876 GEO Accession GSM4689876 SRX8813961 GSM4689877 SRP273093 PRJNA647773 SRS7077797 GSM4689877 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689877 GSM4689877 GEO Accession GSM4689877 SRX8813962 GSM4689878 SRP273093 PRJNA647773 SRS7077798 GSM4689878 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689878 GSM4689878 GEO Accession GSM4689878 SRX8813963 GSM4689879 SRP273093 PRJNA647773 SRS7077799 GSM4689879 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689879 GSM4689879 GEO Accession GSM4689879 SRX8813964 GSM4689880 SRP273093 PRJNA647773 SRS7077801 GSM4689880 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689880 GSM4689880 GEO Accession GSM4689880 SRX8813965 GSM4689881 SRP273093 PRJNA647773 SRS7077800 GSM4689881 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689881 GSM4689881 GEO Accession GSM4689881 SRX8813966 GSM4689882 SRP273093 PRJNA647773 SRS7077802 GSM4689882 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689882 GSM4689882 GEO Accession GSM4689882 SRX8813967 GSM4689883 SRP273093 PRJNA647773 SRS7077804 GSM4689883 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689883 GSM4689883 GEO Accession GSM4689883 SRX8813968 GSM4689884 SRP273093 PRJNA647773 SRS7077803 GSM4689884 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689884 GSM4689884 GEO Accession GSM4689884 SRX8813969 GSM4689885 SRP273093 PRJNA647773 SRS7077806 GSM4689885 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689885 GSM4689885 GEO Accession GSM4689885 SRX8813970 GSM4689886 SRP273093 PRJNA647773 SRS7077805 GSM4689886 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689886 GSM4689886 GEO Accession GSM4689886 SRX8813971 GSM4689887 SRP273093 PRJNA647773 SRS7077807 GSM4689887 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689887 GSM4689887 GEO Accession GSM4689887 SRX8813972 GSM4689888 SRP273093 PRJNA647773 SRS7077808 GSM4689888 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689888 GSM4689888 GEO Accession GSM4689888 SRX8813973 GSM4689889 SRP273093 PRJNA647773 SRS7077810 GSM4689889 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689889 GSM4689889 GEO Accession GSM4689889 SRX8813974 GSM4689890 SRP273093 PRJNA647773 SRS7077809 GSM4689890 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689890 GSM4689890 GEO Accession GSM4689890 SRX8813975 GSM4689891 SRP273093 PRJNA647773 SRS7077812 GSM4689891 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689891 GSM4689891 GEO Accession GSM4689891 SRX8813976 GSM4689892 SRP273093 PRJNA647773 SRS7077811 GSM4689892 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689892 GSM4689892 GEO Accession GSM4689892 SRX8813977 GSM4689893 SRP273093 PRJNA647773 SRS7077814 GSM4689893 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689893 GSM4689893 GEO Accession GSM4689893 SRX8813978 GSM4689894 SRP273093 PRJNA647773 SRS7077813 GSM4689894 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689894 GSM4689894 GEO Accession GSM4689894 SRX8813979 GSM4689895 SRP273093 PRJNA647773 SRS7077815 GSM4689895 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689895 GSM4689895 GEO Accession GSM4689895 SRX8813980 GSM4689896 SRP273093 PRJNA647773 SRS7077816 GSM4689896 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689896 GSM4689896 GEO Accession GSM4689896 SRX8813981 GSM4689897 SRP273093 PRJNA647773 SRS7077817 GSM4689897 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689897 GSM4689897 GEO Accession GSM4689897 SRX8813982 GSM4689898 SRP273093 PRJNA647773 SRS7077818 GSM4689898 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689898 GSM4689898 GEO Accession GSM4689898 SRX8813983 GSM4689899 SRP273093 PRJNA647773 SRS7077819 GSM4689899 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689899 GSM4689899 GEO Accession GSM4689899 SRX8813984 GSM4689900 SRP273093 PRJNA647773 SRS7077821 GSM4689900 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689900 GSM4689900 GEO Accession GSM4689900 SRX8813985 GSM4689901 SRP273093 PRJNA647773 SRS7077820 GSM4689901 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689901 GSM4689901 GEO Accession GSM4689901 SRX8813986 GSM4689902 SRP273093 PRJNA647773 SRS7077823 GSM4689902 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689902 GSM4689902 GEO Accession GSM4689902 SRX8813987 GSM4689903 SRP273093 PRJNA647773 SRS7077822 GSM4689903 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689903 GSM4689903 GEO Accession GSM4689903 SRX8813988 GSM4689904 SRP273093 PRJNA647773 SRS7077824 GSM4689904 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689904 GSM4689904 GEO Accession GSM4689904 SRX8813989 GSM4689905 SRP273093 PRJNA647773 SRS7077825 GSM4689905 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689905 GSM4689905 GEO Accession GSM4689905 SRX8813990 GSM4689906 SRP273093 PRJNA647773 SRS7077827 GSM4689906 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689906 GSM4689906 GEO Accession GSM4689906 SRX8813991 GSM4689907 SRP273093 PRJNA647773 SRS7077826 GSM4689907 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689907 GSM4689907 GEO Accession GSM4689907 SRX8813992 GSM4689908 SRP273093 PRJNA647773 SRS7077828 GSM4689908 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689908 GSM4689908 GEO Accession GSM4689908 SRX8813993 GSM4689909 SRP273093 PRJNA647773 SRS7077829 GSM4689909 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689909 GSM4689909 GEO Accession GSM4689909 SRX8813994 GSM4689910 SRP273093 PRJNA647773 SRS7077830 GSM4689910 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689910 GSM4689910 GEO Accession GSM4689910 SRX8813995 GSM4689911 SRP273093 PRJNA647773 SRS7077831 GSM4689911 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689911 GSM4689911 GEO Accession GSM4689911 SRX8813996 GSM4689912 SRP273093 PRJNA647773 SRS7077832 GSM4689912 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689912 GSM4689912 GEO Accession GSM4689912 SRX8813997 GSM4689913 SRP273093 PRJNA647773 SRS7077833 GSM4689913 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689913 GSM4689913 GEO Accession GSM4689913 SRX8813998 GSM4689914 SRP273093 PRJNA647773 SRS7077834 GSM4689914 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689914 GSM4689914 GEO Accession GSM4689914 SRX8813999 GSM4689915 SRP273093 PRJNA647773 SRS7077836 GSM4689915 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689915 GSM4689915 GEO Accession GSM4689915 SRX8814000 GSM4689916 SRP273093 PRJNA647773 SRS7077835 GSM4689916 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689916 GSM4689916 GEO Accession GSM4689916 SRX8814001 GSM4689917 SRP273093 PRJNA647773 SRS7077838 GSM4689917 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689917 GSM4689917 GEO Accession GSM4689917 SRX8814002 GSM4689918 SRP273093 PRJNA647773 SRS7077837 GSM4689918 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689918 GSM4689918 GEO Accession GSM4689918 SRX8814003 GSM4689919 SRP273093 PRJNA647773 SRS7077839 GSM4689919 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689919 GSM4689919 GEO Accession GSM4689919 SRX8814004 GSM4689920 SRP273093 PRJNA647773 SRS7077840 GSM4689920 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689920 GSM4689920 GEO Accession GSM4689920 SRX8814005 GSM4689921 SRP273093 PRJNA647773 SRS7077841 GSM4689921 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689921 GSM4689921 GEO Accession GSM4689921 SRX8814006 GSM4689922 SRP273093 PRJNA647773 SRS7077842 GSM4689922 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689922 GSM4689922 GEO Accession GSM4689922 SRX8814007 GSM4689923 SRP273093 PRJNA647773 SRS7077843 GSM4689923 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689923 GSM4689923 GEO Accession GSM4689923 SRX8814008 GSM4689924 SRP273093 PRJNA647773 SRS7077844 GSM4689924 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689924 GSM4689924 GEO Accession GSM4689924 SRX8814009 GSM4689925 SRP273093 PRJNA647773 SRS7077845 GSM4689925 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689925 GSM4689925 GEO Accession GSM4689925 SRX8814010 GSM4689926 SRP273093 PRJNA647773 SRS7077846 GSM4689926 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689926 GSM4689926 GEO Accession GSM4689926 SRX8814011 GSM4689927 SRP273093 PRJNA647773 SRS7077847 GSM4689927 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689927 GSM4689927 GEO Accession GSM4689927 SRX8814012 GSM4689928 SRP273093 PRJNA647773 SRS7077848 GSM4689928 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689928 GSM4689928 GEO Accession GSM4689928 SRX8814013 GSM4689929 SRP273093 PRJNA647773 SRS7077850 GSM4689929 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689929 GSM4689929 GEO Accession GSM4689929 SRX8814014 GSM4689930 SRP273093 PRJNA647773 SRS7077849 GSM4689930 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689930 GSM4689930 GEO Accession GSM4689930 SRX8814015 GSM4689931 SRP273093 PRJNA647773 SRS7077851 GSM4689931 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689931 GSM4689931 GEO Accession GSM4689931 SRX8814016 GSM4689932 SRP273093 PRJNA647773 SRS7077852 GSM4689932 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689932 GSM4689932 GEO Accession GSM4689932 SRX8814017 GSM4689933 SRP273093 PRJNA647773 SRS7077854 GSM4689933 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689933 GSM4689933 GEO Accession GSM4689933 SRX8814018 GSM4689934 SRP273093 PRJNA647773 SRS7077855 GSM4689934 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689934 GSM4689934 GEO Accession GSM4689934 SRX8814019 GSM4689935 SRP273093 PRJNA647773 SRS7077853 GSM4689935 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689935 GSM4689935 GEO Accession GSM4689935 SRX8814020 GSM4689936 SRP273093 PRJNA647773 SRS7077856 GSM4689936 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689936 GSM4689936 GEO Accession GSM4689936 SRX8814021 GSM4689937 SRP273093 PRJNA647773 SRS7077857 GSM4689937 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689937 GSM4689937 GEO Accession GSM4689937 SRX8814022 GSM4689938 SRP273093 PRJNA647773 SRS7077858 GSM4689938 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689938 GSM4689938 GEO Accession GSM4689938 SRX8814023 GSM4689939 SRP273093 PRJNA647773 SRS7077859 GSM4689939 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689939 GSM4689939 GEO Accession GSM4689939 SRX8814024 GSM4689940 SRP273093 PRJNA647773 SRS7077860 GSM4689940 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689940 GSM4689940 GEO Accession GSM4689940 SRX8814025 GSM4689941 SRP273093 PRJNA647773 SRS7077861 GSM4689941 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689941 GSM4689941 GEO Accession GSM4689941 SRX8814026 GSM4689942 SRP273093 PRJNA647773 SRS7077862 GSM4689942 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689942 GSM4689942 GEO Accession GSM4689942 SRX8814027 GSM4689943 SRP273093 PRJNA647773 SRS7077863 GSM4689943 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689943 GSM4689943 GEO Accession GSM4689943 SRX8814028 GSM4689944 SRP273093 PRJNA647773 SRS7077864 GSM4689944 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689944 GSM4689944 GEO Accession GSM4689944 SRX8814029 GSM4689945 SRP273093 PRJNA647773 SRS7077865 GSM4689945 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689945 GSM4689945 GEO Accession GSM4689945 SRX8814030 GSM4689946 SRP273093 PRJNA647773 SRS7077866 GSM4689946 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689946 GSM4689946 GEO Accession GSM4689946 SRX8814031 GSM4689947 SRP273093 PRJNA647773 SRS7077867 GSM4689947 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689947 GSM4689947 GEO Accession GSM4689947 SRX8814032 GSM4689948 SRP273093 PRJNA647773 SRS7077868 GSM4689948 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689948 GSM4689948 GEO Accession GSM4689948 SRX8814033 GSM4689949 SRP273093 PRJNA647773 SRS7077869 GSM4689949 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689949 GSM4689949 GEO Accession GSM4689949 SRX8814034 GSM4689950 SRP273093 PRJNA647773 SRS7077870 GSM4689950 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689950 GSM4689950 GEO Accession GSM4689950 SRX8814035 GSM4689951 SRP273093 PRJNA647773 SRS7077871 GSM4689951 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689951 GSM4689951 GEO Accession GSM4689951 SRX8814036 GSM4689952 SRP273093 PRJNA647773 SRS7077872 GSM4689952 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689952 GSM4689952 GEO Accession GSM4689952 SRX8814037 GSM4689953 SRP273093 PRJNA647773 SRS7077873 GSM4689953 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689953 GSM4689953 GEO Accession GSM4689953 SRX8814038 GSM4689954 SRP273093 PRJNA647773 SRS7077874 GSM4689954 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689954 GSM4689954 GEO Accession GSM4689954 SRX8814039 GSM4689955 SRP273093 PRJNA647773 SRS7077876 GSM4689955 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689955 GSM4689955 GEO Accession GSM4689955 SRX8814040 GSM4689956 SRP273093 PRJNA647773 SRS7077875 GSM4689956 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689956 GSM4689956 GEO Accession GSM4689956 SRX8814041 GSM4689957 SRP273093 PRJNA647773 SRS7077878 GSM4689957 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689957 GSM4689957 GEO Accession GSM4689957 SRX8814042 GSM4689958 SRP273093 PRJNA647773 SRS7077877 GSM4689958 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689958 GSM4689958 GEO Accession GSM4689958 SRX8814043 GSM4689959 SRP273093 PRJNA647773 SRS7077879 GSM4689959 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689959 GSM4689959 GEO Accession GSM4689959 SRX8814044 GSM4689960 SRP273093 PRJNA647773 SRS7077880 GSM4689960 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689960 GSM4689960 GEO Accession GSM4689960 SRX8814045 GSM4689961 SRP273093 PRJNA647773 SRS7077881 GSM4689961 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689961 GSM4689961 GEO Accession GSM4689961 SRX8814046 GSM4689962 SRP273093 PRJNA647773 SRS7077882 GSM4689962 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689962 GSM4689962 GEO Accession GSM4689962 SRX8814047 GSM4689963 SRP273093 PRJNA647773 SRS7077883 GSM4689963 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689963 GSM4689963 GEO Accession GSM4689963 SRX8814048 GSM4689964 SRP273093 PRJNA647773 SRS7077885 GSM4689964 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689964 GSM4689964 GEO Accession GSM4689964 SRX8814049 GSM4689965 SRP273093 PRJNA647773 SRS7077884 GSM4689965 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689965 GSM4689965 GEO Accession GSM4689965 SRX8814050 GSM4689966 SRP273093 PRJNA647773 SRS7077887 GSM4689966 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689966 GSM4689966 GEO Accession GSM4689966 SRX8814051 GSM4689967 SRP273093 PRJNA647773 SRS7077886 GSM4689967 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689967 GSM4689967 GEO Accession GSM4689967 SRX8814052 GSM4689968 SRP273093 PRJNA647773 SRS7077888 GSM4689968 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689968 GSM4689968 GEO Accession GSM4689968 SRX8814053 GSM4689969 SRP273093 PRJNA647773 SRS7077889 GSM4689969 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689969 GSM4689969 GEO Accession GSM4689969 SRX8814054 GSM4689970 SRP273093 PRJNA647773 SRS7077890 GSM4689970 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689970 GSM4689970 GEO Accession GSM4689970 SRX8814055 GSM4689971 SRP273093 PRJNA647773 SRS7077891 GSM4689971 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689971 GSM4689971 GEO Accession GSM4689971 SRX8814056 GSM4689972 SRP273093 PRJNA647773 SRS7077892 GSM4689972 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689972 GSM4689972 GEO Accession GSM4689972 SRX8814057 GSM4689973 SRP273093 PRJNA647773 SRS7077893 GSM4689973 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689973 GSM4689973 GEO Accession GSM4689973 SRX8814058 GSM4689974 SRP273093 PRJNA647773 SRS7077894 GSM4689974 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689974 GSM4689974 GEO Accession GSM4689974 SRX8814059 GSM4689975 SRP273093 PRJNA647773 SRS7077896 GSM4689975 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689975 GSM4689975 GEO Accession GSM4689975 SRX8814060 GSM4689976 SRP273093 PRJNA647773 SRS7077895 GSM4689976 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689976 GSM4689976 GEO Accession GSM4689976 SRX8814061 GSM4689977 SRP273093 PRJNA647773 SRS7077898 GSM4689977 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689977 GSM4689977 GEO Accession GSM4689977 SRX8814062 GSM4689978 SRP273093 PRJNA647773 SRS7077897 GSM4689978 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689978 GSM4689978 GEO Accession GSM4689978 SRX8814063 GSM4689979 SRP273093 PRJNA647773 SRS7077899 GSM4689979 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689979 GSM4689979 GEO Accession GSM4689979 SRX8814064 GSM4689980 SRP273093 PRJNA647773 SRS7077900 GSM4689980 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689980 GSM4689980 GEO Accession GSM4689980 SRX8814065 GSM4689981 SRP273093 PRJNA647773 SRS7077901 GSM4689981 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689981 GSM4689981 GEO Accession GSM4689981 SRX8814066 GSM4689982 SRP273093 PRJNA647773 SRS7077902 GSM4689982 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689982 GSM4689982 GEO Accession GSM4689982 SRX8814067 GSM4689983 SRP273093 PRJNA647773 SRS7077903 GSM4689983 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689983 GSM4689983 GEO Accession GSM4689983 SRX8814068 GSM4689984 SRP273093 PRJNA647773 SRS7077904 GSM4689984 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689984 GSM4689984 GEO Accession GSM4689984 SRX8814069 GSM4689985 SRP273093 PRJNA647773 SRS7077905 GSM4689985 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689985 GSM4689985 GEO Accession GSM4689985 SRX8814070 GSM4689986 SRP273093 PRJNA647773 SRS7077906 GSM4689986 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689986 GSM4689986 GEO Accession GSM4689986 SRX8814071 GSM4689987 SRP273093 PRJNA647773 SRS7077907 GSM4689987 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689987 GSM4689987 GEO Accession GSM4689987 SRX8814072 GSM4689988 SRP273093 PRJNA647773 SRS7077909 GSM4689988 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689988 GSM4689988 GEO Accession GSM4689988 SRX8814073 GSM4689989 SRP273093 PRJNA647773 SRS7077908 GSM4689989 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689989 GSM4689989 GEO Accession GSM4689989 SRX8814074 GSM4689990 SRP273093 PRJNA647773 SRS7077911 GSM4689990 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689990 GSM4689990 GEO Accession GSM4689990 SRX8814075 GSM4689991 SRP273093 PRJNA647773 SRS7077910 GSM4689991 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689991 GSM4689991 GEO Accession GSM4689991 SRX8814076 GSM4689992 SRP273093 PRJNA647773 SRS7077913 GSM4689992 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689992 GSM4689992 GEO Accession GSM4689992 SRX8814077 GSM4689993 SRP273093 PRJNA647773 SRS7077912 GSM4689993 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689993 GSM4689993 GEO Accession GSM4689993 SRX8814078 GSM4689994 SRP273093 PRJNA647773 SRS7077914 GSM4689994 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689994 GSM4689994 GEO Accession GSM4689994 SRX8814079 GSM4689995 SRP273093 PRJNA647773 SRS7077915 GSM4689995 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689995 GSM4689995 GEO Accession GSM4689995 SRX8814080 GSM4689996 SRP273093 PRJNA647773 SRS7077917 GSM4689996 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689996 GSM4689996 GEO Accession GSM4689996 SRX8814081 GSM4689997 SRP273093 PRJNA647773 SRS7077916 GSM4689997 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689997 GSM4689997 GEO Accession GSM4689997 SRX8814082 GSM4689998 SRP273093 PRJNA647773 SRS7077919 GSM4689998 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689998 GSM4689998 GEO Accession GSM4689998 SRX8814083 GSM4689999 SRP273093 PRJNA647773 SRS7077918 GSM4689999 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304689999 GSM4689999 GEO Accession GSM4689999 SRX8814084 GSM4690000 SRP273093 PRJNA647773 SRS7077920 GSM4690000 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690000 GSM4690000 GEO Accession GSM4690000 SRX8814085 GSM4690001 SRP273093 PRJNA647773 SRS7077921 GSM4690001 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690001 GSM4690001 GEO Accession GSM4690001 SRX8814086 GSM4690002 SRP273093 PRJNA647773 SRS7077928 GSM4690002 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690002 GSM4690002 GEO Accession GSM4690002 SRX8814087 GSM4690003 SRP273093 PRJNA647773 SRS7077926 GSM4690003 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690003 GSM4690003 GEO Accession GSM4690003 SRX8814088 GSM4690004 SRP273093 PRJNA647773 SRS7077927 GSM4690004 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690004 GSM4690004 GEO Accession GSM4690004 SRX8814089 GSM4690005 SRP273093 PRJNA647773 SRS7077929 GSM4690005 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690005 GSM4690005 GEO Accession GSM4690005 SRX8814090 GSM4690006 SRP273093 PRJNA647773 SRS7077930 GSM4690006 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690006 GSM4690006 GEO Accession GSM4690006 SRX8814091 GSM4690007 SRP273093 PRJNA647773 SRS7077931 GSM4690007 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690007 GSM4690007 GEO Accession GSM4690007 SRX8814092 GSM4690008 SRP273093 PRJNA647773 SRS7077934 GSM4690008 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690008 GSM4690008 GEO Accession GSM4690008 SRX8814093 GSM4690009 SRP273093 PRJNA647773 SRS7077932 GSM4690009 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690009 GSM4690009 GEO Accession GSM4690009 SRX8814094 GSM4690010 SRP273093 PRJNA647773 SRS7077933 GSM4690010 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690010 GSM4690010 GEO Accession GSM4690010 SRX8814095 GSM4690011 SRP273093 PRJNA647773 SRS7077937 GSM4690011 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690011 GSM4690011 GEO Accession GSM4690011 SRX8814096 GSM4690012 SRP273093 PRJNA647773 SRS7077935 GSM4690012 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690012 GSM4690012 GEO Accession GSM4690012 SRX8814097 GSM4690013 SRP273093 PRJNA647773 SRS7077936 GSM4690013 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690013 GSM4690013 GEO Accession GSM4690013 SRX8814098 GSM4690014 SRP273093 PRJNA647773 SRS7077938 GSM4690014 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690014 GSM4690014 GEO Accession GSM4690014 SRX8814099 GSM4690015 SRP273093 PRJNA647773 SRS7077940 GSM4690015 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690015 GSM4690015 GEO Accession GSM4690015 SRX8814100 GSM4690016 SRP273093 PRJNA647773 SRS7077939 GSM4690016 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690016 GSM4690016 GEO Accession GSM4690016 SRX8814101 GSM4690017 SRP273093 PRJNA647773 SRS7077941 GSM4690017 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690017 GSM4690017 GEO Accession GSM4690017 SRX8814102 GSM4690018 SRP273093 PRJNA647773 SRS7077942 GSM4690018 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690018 GSM4690018 GEO Accession GSM4690018 SRX8814103 GSM4690019 SRP273093 PRJNA647773 SRS7077943 GSM4690019 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690019 GSM4690019 GEO Accession GSM4690019 SRX8814104 GSM4690020 SRP273093 PRJNA647773 SRS7077944 GSM4690020 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690020 GSM4690020 GEO Accession GSM4690020 SRX8814105 GSM4690021 SRP273093 PRJNA647773 SRS7077945 GSM4690021 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690021 GSM4690021 GEO Accession GSM4690021 SRX8814106 GSM4690022 SRP273093 PRJNA647773 SRS7077947 GSM4690022 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690022 GSM4690022 GEO Accession GSM4690022 SRX8814107 GSM4690023 SRP273093 PRJNA647773 SRS7077946 GSM4690023 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690023 GSM4690023 GEO Accession GSM4690023 SRX8814108 GSM4690024 SRP273093 PRJNA647773 SRS7077948 GSM4690024 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690024 GSM4690024 GEO Accession GSM4690024 SRX8814109 GSM4690025 SRP273093 PRJNA647773 SRS7077949 GSM4690025 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690025 GSM4690025 GEO Accession GSM4690025 SRX8814110 GSM4690026 SRP273093 PRJNA647773 SRS7077950 GSM4690026 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690026 GSM4690026 GEO Accession GSM4690026 SRX8814111 GSM4690027 SRP273093 PRJNA647773 SRS7077951 GSM4690027 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690027 GSM4690027 GEO Accession GSM4690027 SRX8814112 GSM4690028 SRP273093 PRJNA647773 SRS7077953 GSM4690028 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690028 GSM4690028 GEO Accession GSM4690028 SRX8814113 GSM4690029 SRP273093 PRJNA647773 SRS7077952 GSM4690029 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690029 GSM4690029 GEO Accession GSM4690029 SRX8814114 GSM4690030 SRP273093 PRJNA647773 SRS7077954 GSM4690030 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690030 GSM4690030 GEO Accession GSM4690030 SRX8814115 GSM4690031 SRP273093 PRJNA647773 SRS7077955 GSM4690031 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690031 GSM4690031 GEO Accession GSM4690031 SRX8814116 GSM4690032 SRP273093 PRJNA647773 SRS7077956 GSM4690032 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690032 GSM4690032 GEO Accession GSM4690032 SRX8814117 GSM4690033 SRP273093 PRJNA647773 SRS7077957 GSM4690033 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690033 GSM4690033 GEO Accession GSM4690033 SRX8814118 GSM4690034 SRP273093 PRJNA647773 SRS7077958 GSM4690034 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690034 GSM4690034 GEO Accession GSM4690034 SRX8814119 GSM4690035 SRP273093 PRJNA647773 SRS7077959 GSM4690035 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690035 GSM4690035 GEO Accession GSM4690035 SRX8814120 GSM4690036 SRP273093 PRJNA647773 SRS7077960 GSM4690036 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690036 GSM4690036 GEO Accession GSM4690036 SRX8814121 GSM4690037 SRP273093 PRJNA647773 SRS7077961 GSM4690037 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690037 GSM4690037 GEO Accession GSM4690037 SRX8814122 GSM4690038 SRP273093 PRJNA647773 SRS7077963 GSM4690038 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690038 GSM4690038 GEO Accession GSM4690038 SRX8814123 GSM4690039 SRP273093 PRJNA647773 SRS7077962 GSM4690039 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690039 GSM4690039 GEO Accession GSM4690039 SRX8814124 GSM4690040 SRP273093 PRJNA647773 SRS7077964 GSM4690040 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690040 GSM4690040 GEO Accession GSM4690040 SRX8814125 GSM4690041 SRP273093 PRJNA647773 SRS7077965 GSM4690041 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690041 GSM4690041 GEO Accession GSM4690041 SRX8814126 GSM4690042 SRP273093 PRJNA647773 SRS7077968 GSM4690042 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690042 GSM4690042 GEO Accession GSM4690042 SRX8814127 GSM4690043 SRP273093 PRJNA647773 SRS7077966 GSM4690043 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690043 GSM4690043 GEO Accession GSM4690043 SRX8814128 GSM4690044 SRP273093 PRJNA647773 SRS7077967 GSM4690044 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690044 GSM4690044 GEO Accession GSM4690044 SRX8814129 GSM4690045 SRP273093 PRJNA647773 SRS7077969 GSM4690045 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690045 GSM4690045 GEO Accession GSM4690045 SRX8814130 GSM4690046 SRP273093 PRJNA647773 SRS7077971 GSM4690046 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690046 GSM4690046 GEO Accession GSM4690046 SRX8814131 GSM4690047 SRP273093 PRJNA647773 SRS7077970 GSM4690047 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690047 GSM4690047 GEO Accession GSM4690047 SRX8814132 GSM4690048 SRP273093 PRJNA647773 SRS7077972 GSM4690048 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690048 GSM4690048 GEO Accession GSM4690048 SRX8814133 GSM4690049 SRP273093 PRJNA647773 SRS7077973 GSM4690049 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690049 GSM4690049 GEO Accession GSM4690049 SRX8814134 GSM4690050 SRP273093 PRJNA647773 SRS7077974 GSM4690050 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690050 GSM4690050 GEO Accession GSM4690050 SRX8814135 GSM4690051 SRP273093 PRJNA647773 SRS7077975 GSM4690051 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690051 GSM4690051 GEO Accession GSM4690051 SRX8814136 GSM4690052 SRP273093 PRJNA647773 SRS7077976 GSM4690052 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690052 GSM4690052 GEO Accession GSM4690052 SRX8814137 GSM4690053 SRP273093 PRJNA647773 SRS7077977 GSM4690053 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690053 GSM4690053 GEO Accession GSM4690053 SRX8814138 GSM4690054 SRP273093 PRJNA647773 SRS7076747 GSM4690054 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690054 GSM4690054 GEO Accession GSM4690054 SRX8814139 GSM4690055 SRP273093 PRJNA647773 SRS7077978 GSM4690055 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690055 GSM4690055 GEO Accession GSM4690055 SRX8814140 GSM4690056 SRP273093 PRJNA647773 SRS7077979 GSM4690056 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690056 GSM4690056 GEO Accession GSM4690056 SRX8814141 GSM4690057 SRP273093 PRJNA647773 SRS7077980 GSM4690057 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690057 GSM4690057 GEO Accession GSM4690057 SRX8814142 GSM4690058 SRP273093 PRJNA647773 SRS7077981 GSM4690058 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690058 GSM4690058 GEO Accession GSM4690058 SRX8814143 GSM4690059 SRP273093 PRJNA647773 SRS7077982 GSM4690059 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690059 GSM4690059 GEO Accession GSM4690059 SRX8814144 GSM4690060 SRP273093 PRJNA647773 SRS7077983 GSM4690060 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690060 GSM4690060 GEO Accession GSM4690060 SRX8814145 GSM4690061 SRP273093 PRJNA647773 SRS7077984 GSM4690061 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690061 GSM4690061 GEO Accession GSM4690061 SRX8814146 GSM4690062 SRP273093 PRJNA647773 SRS7077985 GSM4690062 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690062 GSM4690062 GEO Accession GSM4690062 SRX8814147 GSM4690063 SRP273093 PRJNA647773 SRS7077986 GSM4690063 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690063 GSM4690063 GEO Accession GSM4690063 SRX8814148 GSM4690064 SRP273093 PRJNA647773 SRS7077987 GSM4690064 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690064 GSM4690064 GEO Accession GSM4690064 SRX8814149 GSM4690065 SRP273093 PRJNA647773 SRS7077988 GSM4690065 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690065 GSM4690065 GEO Accession GSM4690065 SRX8814150 GSM4690066 SRP273093 PRJNA647773 SRS7077989 GSM4690066 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690066 GSM4690066 GEO Accession GSM4690066 SRX8814151 GSM4690067 SRP273093 PRJNA647773 SRS7077990 GSM4690067 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690067 GSM4690067 GEO Accession GSM4690067 SRX8814152 GSM4690068 SRP273093 PRJNA647773 SRS7077991 GSM4690068 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690068 GSM4690068 GEO Accession GSM4690068 SRX8814153 GSM4690069 SRP273093 PRJNA647773 SRS7077992 GSM4690069 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690069 GSM4690069 GEO Accession GSM4690069 SRX8814154 GSM4690070 SRP273093 PRJNA647773 SRS7077993 GSM4690070 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690070 GSM4690070 GEO Accession GSM4690070 SRX8814155 GSM4690071 SRP273093 PRJNA647773 SRS7077994 GSM4690071 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690071 GSM4690071 GEO Accession GSM4690071 SRX8814156 GSM4690072 SRP273093 PRJNA647773 SRS7077995 GSM4690072 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690072 GSM4690072 GEO Accession GSM4690072 SRX8814157 GSM4690073 SRP273093 PRJNA647773 SRS7077996 GSM4690073 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690073 GSM4690073 GEO Accession GSM4690073 SRX8814158 GSM4690074 SRP273093 PRJNA647773 SRS7077997 GSM4690074 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690074 GSM4690074 GEO Accession GSM4690074 SRX8814159 GSM4690075 SRP273093 PRJNA647773 SRS7077999 GSM4690075 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690075 GSM4690075 GEO Accession GSM4690075 SRX8814160 GSM4690076 SRP273093 PRJNA647773 SRS7077998 GSM4690076 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690076 GSM4690076 GEO Accession GSM4690076 SRX8814161 GSM4690077 SRP273093 PRJNA647773 SRS7078000 GSM4690077 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690077 GSM4690077 GEO Accession GSM4690077 SRX8814162 GSM4690078 SRP273093 PRJNA647773 SRS7078001 GSM4690078 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690078 GSM4690078 GEO Accession GSM4690078 SRX8814163 GSM4690079 SRP273093 PRJNA647773 SRS7078003 GSM4690079 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690079 GSM4690079 GEO Accession GSM4690079 SRX8814164 GSM4690080 SRP273093 PRJNA647773 SRS7078002 GSM4690080 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690080 GSM4690080 GEO Accession GSM4690080 SRX8814165 GSM4690081 SRP273093 PRJNA647773 SRS7078005 GSM4690081 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690081 GSM4690081 GEO Accession GSM4690081 SRX8814166 GSM4690082 SRP273093 PRJNA647773 SRS7078004 GSM4690082 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690082 GSM4690082 GEO Accession GSM4690082 SRX8814167 GSM4690083 SRP273093 PRJNA647773 SRS7078007 GSM4690083 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690083 GSM4690083 GEO Accession GSM4690083 SRX8814168 GSM4690084 SRP273093 PRJNA647773 SRS7078006 GSM4690084 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690084 GSM4690084 GEO Accession GSM4690084 SRX8814169 GSM4690085 SRP273093 PRJNA647773 SRS7078008 GSM4690085 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690085 GSM4690085 GEO Accession GSM4690085 SRX8814170 GSM4690086 SRP273093 PRJNA647773 SRS7078009 GSM4690086 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690086 GSM4690086 GEO Accession GSM4690086 SRX8814171 GSM4690087 SRP273093 PRJNA647773 SRS7078010 GSM4690087 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690087 GSM4690087 GEO Accession GSM4690087 SRX8814172 GSM4690088 SRP273093 PRJNA647773 SRS7078011 GSM4690088 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690088 GSM4690088 GEO Accession GSM4690088 SRX8814173 GSM4690089 SRP273093 PRJNA647773 SRS7078012 GSM4690089 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690089 GSM4690089 GEO Accession GSM4690089 SRX8814174 GSM4690090 SRP273093 PRJNA647773 SRS7078013 GSM4690090 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690090 GSM4690090 GEO Accession GSM4690090 SRX8814175 GSM4690091 SRP273093 PRJNA647773 SRS7078014 GSM4690091 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690091 GSM4690091 GEO Accession GSM4690091 SRX8814176 GSM4690092 SRP273093 PRJNA647773 SRS7078015 GSM4690092 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690092 GSM4690092 GEO Accession GSM4690092 SRX8814177 GSM4690093 SRP273093 PRJNA647773 SRS7078016 GSM4690093 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690093 GSM4690093 GEO Accession GSM4690093 SRX8814178 GSM4690094 SRP273093 PRJNA647773 SRS7078018 GSM4690094 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690094 GSM4690094 GEO Accession GSM4690094 SRX8814179 GSM4690095 SRP273093 PRJNA647773 SRS7078017 GSM4690095 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690095 GSM4690095 GEO Accession GSM4690095 SRX8814180 GSM4690096 SRP273093 PRJNA647773 SRS7078019 GSM4690096 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690096 GSM4690096 GEO Accession GSM4690096 SRX8814181 GSM4690097 SRP273093 PRJNA647773 SRS7078020 GSM4690097 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690097 GSM4690097 GEO Accession GSM4690097 SRX8814182 GSM4690098 SRP273093 PRJNA647773 SRS7078021 GSM4690098 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690098 GSM4690098 GEO Accession GSM4690098 SRX8814183 GSM4690099 SRP273093 PRJNA647773 SRS7078022 GSM4690099 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690099 GSM4690099 GEO Accession GSM4690099 SRX8814184 GSM4690100 SRP273093 PRJNA647773 SRS7078023 GSM4690100 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690100 GSM4690100 GEO Accession GSM4690100 SRX8814185 GSM4690101 SRP273093 PRJNA647773 SRS7078024 GSM4690101 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690101 GSM4690101 GEO Accession GSM4690101 SRX8814186 GSM4690102 SRP273093 PRJNA647773 SRS7078025 GSM4690102 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690102 GSM4690102 GEO Accession GSM4690102 SRX8814187 GSM4690103 SRP273093 PRJNA647773 SRS7078027 GSM4690103 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690103 GSM4690103 GEO Accession GSM4690103 SRX8814188 GSM4690104 SRP273093 PRJNA647773 SRS7078026 GSM4690104 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690104 GSM4690104 GEO Accession GSM4690104 SRX8814189 GSM4690105 SRP273093 PRJNA647773 SRS7078028 GSM4690105 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690105 GSM4690105 GEO Accession GSM4690105 SRX8814190 GSM4690106 SRP273093 PRJNA647773 SRS7078029 GSM4690106 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690106 GSM4690106 GEO Accession GSM4690106 SRX8814191 GSM4690107 SRP273093 PRJNA647773 SRS7078030 GSM4690107 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690107 GSM4690107 GEO Accession GSM4690107 SRX8814192 GSM4690108 SRP273093 PRJNA647773 SRS7078031 GSM4690108 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690108 GSM4690108 GEO Accession GSM4690108 SRX8814193 GSM4690109 SRP273093 PRJNA647773 SRS7078032 GSM4690109 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690109 GSM4690109 GEO Accession GSM4690109 SRX8814194 GSM4690110 SRP273093 PRJNA647773 SRS7078033 GSM4690110 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690110 GSM4690110 GEO Accession GSM4690110 SRX8814195 GSM4690111 SRP273093 PRJNA647773 SRS7078034 GSM4690111 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690111 GSM4690111 GEO Accession GSM4690111 SRX8814196 GSM4690112 SRP273093 PRJNA647773 SRS7078035 GSM4690112 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690112 GSM4690112 GEO Accession GSM4690112 SRX8814197 GSM4690113 SRP273093 PRJNA647773 SRS7078037 GSM4690113 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690113 GSM4690113 GEO Accession GSM4690113 SRX8814198 GSM4690114 SRP273093 PRJNA647773 SRS7078036 GSM4690114 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690114 GSM4690114 GEO Accession GSM4690114 SRX8814199 GSM4690115 SRP273093 PRJNA647773 SRS7078038 GSM4690115 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690115 GSM4690115 GEO Accession GSM4690115 SRX8814200 GSM4690116 SRP273093 PRJNA647773 SRS7078039 GSM4690116 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690116 GSM4690116 GEO Accession GSM4690116 SRX8814201 GSM4690117 SRP273093 PRJNA647773 SRS7078040 GSM4690117 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690117 GSM4690117 GEO Accession GSM4690117 SRX8814202 GSM4690118 SRP273093 PRJNA647773 SRS7078041 GSM4690118 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690118 GSM4690118 GEO Accession GSM4690118 SRX8814203 GSM4690119 SRP273093 PRJNA647773 SRS7078042 GSM4690119 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690119 GSM4690119 GEO Accession GSM4690119 SRX8814204 GSM4690120 SRP273093 PRJNA647773 SRS7078043 GSM4690120 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690120 GSM4690120 GEO Accession GSM4690120 SRX8814205 GSM4690121 SRP273093 PRJNA647773 SRS7078044 GSM4690121 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690121 GSM4690121 GEO Accession GSM4690121 SRX8814206 GSM4690122 SRP273093 PRJNA647773 SRS7078045 GSM4690122 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690122 GSM4690122 GEO Accession GSM4690122 SRX8814207 GSM4690123 SRP273093 PRJNA647773 SRS7078047 GSM4690123 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690123 GSM4690123 GEO Accession GSM4690123 SRX8814208 GSM4690124 SRP273093 PRJNA647773 SRS7078046 GSM4690124 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690124 GSM4690124 GEO Accession GSM4690124 SRX8814209 GSM4690125 SRP273093 PRJNA647773 SRS7078048 GSM4690125 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690125 GSM4690125 GEO Accession GSM4690125 SRX8814210 GSM4690126 SRP273093 PRJNA647773 SRS7078050 GSM4690126 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690126 GSM4690126 GEO Accession GSM4690126 SRX8814211 GSM4690127 SRP273093 PRJNA647773 SRS7078049 GSM4690127 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690127 GSM4690127 GEO Accession GSM4690127 SRX8814212 GSM4690128 SRP273093 PRJNA647773 SRS7078051 GSM4690128 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690128 GSM4690128 GEO Accession GSM4690128 SRX8814213 GSM4690129 SRP273093 PRJNA647773 SRS7078052 GSM4690129 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690129 GSM4690129 GEO Accession GSM4690129 SRX8814214 GSM4690130 SRP273093 PRJNA647773 SRS7078053 GSM4690130 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690130 GSM4690130 GEO Accession GSM4690130 SRX8814215 GSM4690131 SRP273093 PRJNA647773 SRS7078056 GSM4690131 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690131 GSM4690131 GEO Accession GSM4690131 SRX8814216 GSM4690132 SRP273093 PRJNA647773 SRS7078054 GSM4690132 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690132 GSM4690132 GEO Accession GSM4690132 SRX8814217 GSM4690133 SRP273093 PRJNA647773 SRS7078055 GSM4690133 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690133 GSM4690133 GEO Accession GSM4690133 SRX8814218 GSM4690134 SRP273093 PRJNA647773 SRS7078057 GSM4690134 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690134 GSM4690134 GEO Accession GSM4690134 SRX8814219 GSM4690135 SRP273093 PRJNA647773 SRS7078058 GSM4690135 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690135 GSM4690135 GEO Accession GSM4690135 SRX8814220 GSM4690136 SRP273093 PRJNA647773 SRS7078059 GSM4690136 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690136 GSM4690136 GEO Accession GSM4690136 SRX8814221 GSM4690137 SRP273093 PRJNA647773 SRS7078060 GSM4690137 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690137 GSM4690137 GEO Accession GSM4690137 SRX8814222 GSM4690138 SRP273093 PRJNA647773 SRS7078062 GSM4690138 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690138 GSM4690138 GEO Accession GSM4690138 SRX8814223 GSM4690139 SRP273093 PRJNA647773 SRS7078061 GSM4690139 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690139 GSM4690139 GEO Accession GSM4690139 SRX8814224 GSM4690140 SRP273093 PRJNA647773 SRS7078063 GSM4690140 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690140 GSM4690140 GEO Accession GSM4690140 SRX8814225 GSM4690141 SRP273093 PRJNA647773 SRS7078064 GSM4690141 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690141 GSM4690141 GEO Accession GSM4690141 SRX8814226 GSM4690142 SRP273093 PRJNA647773 SRS7078065 GSM4690142 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690142 GSM4690142 GEO Accession GSM4690142 SRX8814227 GSM4690143 SRP273093 PRJNA647773 SRS7078066 GSM4690143 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690143 GSM4690143 GEO Accession GSM4690143 SRX8814228 GSM4690144 SRP273093 PRJNA647773 SRS7078068 GSM4690144 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690144 GSM4690144 GEO Accession GSM4690144 SRX8814229 GSM4690145 SRP273093 PRJNA647773 SRS7078067 GSM4690145 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690145 GSM4690145 GEO Accession GSM4690145 SRX8814230 GSM4690146 SRP273093 PRJNA647773 SRS7078069 GSM4690146 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690146 GSM4690146 GEO Accession GSM4690146 SRX8814231 GSM4690147 SRP273093 PRJNA647773 SRS7078070 GSM4690147 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690147 GSM4690147 GEO Accession GSM4690147 SRX8814232 GSM4690148 SRP273093 PRJNA647773 SRS7078072 GSM4690148 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690148 GSM4690148 GEO Accession GSM4690148 SRX8814233 GSM4690149 SRP273093 PRJNA647773 SRS7078071 GSM4690149 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690149 GSM4690149 GEO Accession GSM4690149 SRX8814234 GSM4690150 SRP273093 PRJNA647773 SRS7078073 GSM4690150 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690150 GSM4690150 GEO Accession GSM4690150 SRX8814235 GSM4690151 SRP273093 PRJNA647773 SRS7078074 GSM4690151 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690151 GSM4690151 GEO Accession GSM4690151 SRX8814236 GSM4690152 SRP273093 PRJNA647773 SRS7078075 GSM4690152 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690152 GSM4690152 GEO Accession GSM4690152 SRX8814237 GSM4690153 SRP273093 PRJNA647773 SRS7078076 GSM4690153 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690153 GSM4690153 GEO Accession GSM4690153 SRX8814238 GSM4690154 SRP273093 PRJNA647773 SRS7078077 GSM4690154 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690154 GSM4690154 GEO Accession GSM4690154 SRX8814239 GSM4690155 SRP273093 PRJNA647773 SRS7078078 GSM4690155 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690155 GSM4690155 GEO Accession GSM4690155 SRX8814240 GSM4690156 SRP273093 PRJNA647773 SRS7078079 GSM4690156 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690156 GSM4690156 GEO Accession GSM4690156 SRX8814241 GSM4690157 SRP273093 PRJNA647773 SRS7078081 GSM4690157 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690157 GSM4690157 GEO Accession GSM4690157 SRX8814242 GSM4690158 SRP273093 PRJNA647773 SRS7078080 GSM4690158 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690158 GSM4690158 GEO Accession GSM4690158 SRX8814243 GSM4690159 SRP273093 PRJNA647773 SRS7078082 GSM4690159 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690159 GSM4690159 GEO Accession GSM4690159 SRX8814244 GSM4690160 SRP273093 PRJNA647773 SRS7078085 GSM4690160 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690160 GSM4690160 GEO Accession GSM4690160 SRX8814245 GSM4690161 SRP273093 PRJNA647773 SRS7078084 GSM4690161 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690161 GSM4690161 GEO Accession GSM4690161 SRX8814246 GSM4690162 SRP273093 PRJNA647773 SRS7078083 GSM4690162 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690162 GSM4690162 GEO Accession GSM4690162 SRX8814247 GSM4690163 SRP273093 PRJNA647773 SRS7078087 GSM4690163 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690163 GSM4690163 GEO Accession GSM4690163 SRX8814248 GSM4690164 SRP273093 PRJNA647773 SRS7078088 GSM4690164 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690164 GSM4690164 GEO Accession GSM4690164 SRX8814249 GSM4690165 SRP273093 PRJNA647773 SRS7078089 GSM4690165 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690165 GSM4690165 GEO Accession GSM4690165 SRX8814250 GSM4690166 SRP273093 PRJNA647773 SRS7078086 GSM4690166 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690166 GSM4690166 GEO Accession GSM4690166 SRX8814251 GSM4690167 SRP273093 PRJNA647773 SRS7078090 GSM4690167 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690167 GSM4690167 GEO Accession GSM4690167 SRX8814252 GSM4690168 SRP273093 PRJNA647773 SRS7078091 GSM4690168 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690168 GSM4690168 GEO Accession GSM4690168 SRX8814253 GSM4690169 SRP273093 PRJNA647773 SRS7078093 GSM4690169 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690169 GSM4690169 GEO Accession GSM4690169 SRX8814254 GSM4690170 SRP273093 PRJNA647773 SRS7078092 GSM4690170 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690170 GSM4690170 GEO Accession GSM4690170 SRX8814255 GSM4690171 SRP273093 PRJNA647773 SRS7078096 GSM4690171 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690171 GSM4690171 GEO Accession GSM4690171 SRX8814256 GSM4690172 SRP273093 PRJNA647773 SRS7078094 GSM4690172 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690172 GSM4690172 GEO Accession GSM4690172 SRX8814257 GSM4690173 SRP273093 PRJNA647773 SRS7078095 GSM4690173 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690173 GSM4690173 GEO Accession GSM4690173 SRX8814258 GSM4690174 SRP273093 PRJNA647773 SRS7078098 GSM4690174 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690174 GSM4690174 GEO Accession GSM4690174 SRX8814259 GSM4690175 SRP273093 PRJNA647773 SRS7078097 GSM4690175 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690175 GSM4690175 GEO Accession GSM4690175 SRX8814260 GSM4690176 SRP273093 PRJNA647773 SRS7078100 GSM4690176 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690176 GSM4690176 GEO Accession GSM4690176 SRX8814261 GSM4690177 SRP273093 PRJNA647773 SRS7078099 GSM4690177 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690177 GSM4690177 GEO Accession GSM4690177 SRX8814262 GSM4690178 SRP273093 PRJNA647773 SRS7078102 GSM4690178 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690178 GSM4690178 GEO Accession GSM4690178 SRX8814263 GSM4690179 SRP273093 PRJNA647773 SRS7078101 GSM4690179 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690179 GSM4690179 GEO Accession GSM4690179 SRX8814264 GSM4690180 SRP273093 PRJNA647773 SRS7078104 GSM4690180 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690180 GSM4690180 GEO Accession GSM4690180 SRX8814265 GSM4690181 SRP273093 PRJNA647773 SRS7078103 GSM4690181 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690181 GSM4690181 GEO Accession GSM4690181 SRX8814266 GSM4690182 SRP273093 PRJNA647773 SRS7078105 GSM4690182 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690182 GSM4690182 GEO Accession GSM4690182 SRX8814267 GSM4690183 SRP273093 PRJNA647773 SRS7078107 GSM4690183 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690183 GSM4690183 GEO Accession GSM4690183 SRX8814268 GSM4690184 SRP273093 PRJNA647773 SRS7078106 GSM4690184 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690184 GSM4690184 GEO Accession GSM4690184 SRX8814269 GSM4690185 SRP273093 PRJNA647773 SRS7078108 GSM4690185 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690185 GSM4690185 GEO Accession GSM4690185 SRX8814270 GSM4690186 SRP273093 PRJNA647773 SRS7078109 GSM4690186 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690186 GSM4690186 GEO Accession GSM4690186 SRX8814271 GSM4690187 SRP273093 PRJNA647773 SRS7078112 GSM4690187 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690187 GSM4690187 GEO Accession GSM4690187 SRX8814272 GSM4690188 SRP273093 PRJNA647773 SRS7078110 GSM4690188 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690188 GSM4690188 GEO Accession GSM4690188 SRX8814273 GSM4690189 SRP273093 PRJNA647773 SRS7078111 GSM4690189 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690189 GSM4690189 GEO Accession GSM4690189 SRX8814274 GSM4690190 SRP273093 PRJNA647773 SRS7078113 GSM4690190 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690190 GSM4690190 GEO Accession GSM4690190 SRX8814275 GSM4690191 SRP273093 PRJNA647773 SRS7078114 GSM4690191 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690191 GSM4690191 GEO Accession GSM4690191 SRX8814276 GSM4690192 SRP273093 PRJNA647773 SRS7078115 GSM4690192 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690192 GSM4690192 GEO Accession GSM4690192 SRX8814277 GSM4690193 SRP273093 PRJNA647773 SRS7078116 GSM4690193 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690193 GSM4690193 GEO Accession GSM4690193 SRX8814278 GSM4690194 SRP273093 PRJNA647773 SRS7078117 GSM4690194 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690194 GSM4690194 GEO Accession GSM4690194 SRX8814279 GSM4690195 SRP273093 PRJNA647773 SRS7078118 GSM4690195 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690195 GSM4690195 GEO Accession GSM4690195 SRX8814280 GSM4690196 SRP273093 PRJNA647773 SRS7078119 GSM4690196 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690196 GSM4690196 GEO Accession GSM4690196 SRX8814281 GSM4690197 SRP273093 PRJNA647773 SRS7078120 GSM4690197 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690197 GSM4690197 GEO Accession GSM4690197 SRX8814282 GSM4690198 SRP273093 PRJNA647773 SRS7078121 GSM4690198 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690198 GSM4690198 GEO Accession GSM4690198 SRX8814283 GSM4690199 SRP273093 PRJNA647773 SRS7078122 GSM4690199 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690199 GSM4690199 GEO Accession GSM4690199 SRX8814284 GSM4690200 SRP273093 PRJNA647773 SRS7078123 GSM4690200 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690200 GSM4690200 GEO Accession GSM4690200 SRX8814285 GSM4690201 SRP273093 PRJNA647773 SRS7078124 GSM4690201 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690201 GSM4690201 GEO Accession GSM4690201 SRX8814286 GSM4690202 SRP273093 PRJNA647773 SRS7078125 GSM4690202 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690202 GSM4690202 GEO Accession GSM4690202 SRX8814287 GSM4690203 SRP273093 PRJNA647773 SRS7078127 GSM4690203 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690203 GSM4690203 GEO Accession GSM4690203 SRX8814288 GSM4690204 SRP273093 PRJNA647773 SRS7078126 GSM4690204 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690204 GSM4690204 GEO Accession GSM4690204 SRX8814289 GSM4690205 SRP273093 PRJNA647773 SRS7078128 GSM4690205 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690205 GSM4690205 GEO Accession GSM4690205 SRX8814290 GSM4690206 SRP273093 PRJNA647773 SRS7078129 GSM4690206 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690206 GSM4690206 GEO Accession GSM4690206 SRX8814291 GSM4690207 SRP273093 PRJNA647773 SRS7078130 GSM4690207 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690207 GSM4690207 GEO Accession GSM4690207 SRX8814292 GSM4690208 SRP273093 PRJNA647773 SRS7078131 GSM4690208 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690208 GSM4690208 GEO Accession GSM4690208 SRX8814293 GSM4690209 SRP273093 PRJNA647773 SRS7078132 GSM4690209 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690209 GSM4690209 GEO Accession GSM4690209 SRX8814294 GSM4690210 SRP273093 PRJNA647773 SRS7078133 GSM4690210 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690210 GSM4690210 GEO Accession GSM4690210 SRX8814295 GSM4690211 SRP273093 PRJNA647773 SRS7078134 GSM4690211 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690211 GSM4690211 GEO Accession GSM4690211 SRX8814296 GSM4690212 SRP273093 PRJNA647773 SRS7078136 GSM4690212 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690212 GSM4690212 GEO Accession GSM4690212 SRX8814297 GSM4690213 SRP273093 PRJNA647773 SRS7078135 GSM4690213 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690213 GSM4690213 GEO Accession GSM4690213 SRX8814298 GSM4690214 SRP273093 PRJNA647773 SRS7078137 GSM4690214 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690214 GSM4690214 GEO Accession GSM4690214 SRX8814299 GSM4690215 SRP273093 PRJNA647773 SRS7078138 GSM4690215 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690215 GSM4690215 GEO Accession GSM4690215 SRX8814300 GSM4690216 SRP273093 PRJNA647773 SRS7078139 GSM4690216 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690216 GSM4690216 GEO Accession GSM4690216 SRX8814301 GSM4690217 SRP273093 PRJNA647773 SRS7078140 GSM4690217 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690217 GSM4690217 GEO Accession GSM4690217 SRX8814302 GSM4690218 SRP273093 PRJNA647773 SRS7078141 GSM4690218 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690218 GSM4690218 GEO Accession GSM4690218 SRX8814303 GSM4690219 SRP273093 PRJNA647773 SRS7078142 GSM4690219 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690219 GSM4690219 GEO Accession GSM4690219 SRX8814304 GSM4690220 SRP273093 PRJNA647773 SRS7078143 GSM4690220 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690220 GSM4690220 GEO Accession GSM4690220 SRX8814305 GSM4690221 SRP273093 PRJNA647773 SRS7078144 GSM4690221 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690221 GSM4690221 GEO Accession GSM4690221 SRX8814306 GSM4690222 SRP273093 PRJNA647773 SRS7078145 GSM4690222 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690222 GSM4690222 GEO Accession GSM4690222 SRX8814307 GSM4690223 SRP273093 PRJNA647773 SRS7078146 GSM4690223 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690223 GSM4690223 GEO Accession GSM4690223 SRX8814308 GSM4690224 SRP273093 PRJNA647773 SRS7078147 GSM4690224 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690224 GSM4690224 GEO Accession GSM4690224 SRX8814309 GSM4690225 SRP273093 PRJNA647773 SRS7078148 GSM4690225 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690225 GSM4690225 GEO Accession GSM4690225 SRX8814310 GSM4690226 SRP273093 PRJNA647773 SRS7078149 GSM4690226 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690226 GSM4690226 GEO Accession GSM4690226 SRX8814311 GSM4690227 SRP273093 PRJNA647773 SRS7078151 GSM4690227 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690227 GSM4690227 GEO Accession GSM4690227 SRX8814312 GSM4690228 SRP273093 PRJNA647773 SRS7078150 GSM4690228 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690228 GSM4690228 GEO Accession GSM4690228 SRX8814313 GSM4690229 SRP273093 PRJNA647773 SRS7078174 GSM4690229 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690229 GSM4690229 GEO Accession GSM4690229 SRX8814314 GSM4690230 SRP273093 PRJNA647773 SRS7078152 GSM4690230 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690230 GSM4690230 GEO Accession GSM4690230 SRX8814315 GSM4690231 SRP273093 PRJNA647773 SRS7078153 GSM4690231 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690231 GSM4690231 GEO Accession GSM4690231 SRX8814316 GSM4690232 SRP273093 PRJNA647773 SRS7078154 GSM4690232 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690232 GSM4690232 GEO Accession GSM4690232 SRX8814317 GSM4690233 SRP273093 PRJNA647773 SRS7078155 GSM4690233 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690233 GSM4690233 GEO Accession GSM4690233 SRX8814318 GSM4690234 SRP273093 PRJNA647773 SRS7078156 GSM4690234 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690234 GSM4690234 GEO Accession GSM4690234 SRX8814319 GSM4690235 SRP273093 PRJNA647773 SRS7078161 GSM4690235 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690235 GSM4690235 GEO Accession GSM4690235 SRX8814320 GSM4690236 SRP273093 PRJNA647773 SRS7078157 GSM4690236 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690236 GSM4690236 GEO Accession GSM4690236 SRX8814321 GSM4690237 SRP273093 PRJNA647773 SRS7078158 GSM4690237 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690237 GSM4690237 GEO Accession GSM4690237 SRX8814322 GSM4690238 SRP273093 PRJNA647773 SRS7078159 GSM4690238 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690238 GSM4690238 GEO Accession GSM4690238 SRX8814323 GSM4690239 SRP273093 PRJNA647773 SRS7078162 GSM4690239 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690239 GSM4690239 GEO Accession GSM4690239 SRX8814324 GSM4690240 SRP273093 PRJNA647773 SRS7078163 GSM4690240 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690240 GSM4690240 GEO Accession GSM4690240 SRX8814325 GSM4690241 SRP273093 PRJNA647773 SRS7078160 GSM4690241 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690241 GSM4690241 GEO Accession GSM4690241 SRX8814326 GSM4690242 SRP273093 PRJNA647773 SRS7078164 GSM4690242 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690242 GSM4690242 GEO Accession GSM4690242 SRX8814327 GSM4690243 SRP273093 PRJNA647773 SRS7078165 GSM4690243 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690243 GSM4690243 GEO Accession GSM4690243 SRX8814328 GSM4690244 SRP273093 PRJNA647773 SRS7078166 GSM4690244 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690244 GSM4690244 GEO Accession GSM4690244 SRX8814329 GSM4690245 SRP273093 PRJNA647773 SRS7078167 GSM4690245 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690245 GSM4690245 GEO Accession GSM4690245 SRX8814330 GSM4690246 SRP273093 PRJNA647773 SRS7078168 GSM4690246 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690246 GSM4690246 GEO Accession GSM4690246 SRX8814331 GSM4690247 SRP273093 PRJNA647773 SRS7078169 GSM4690247 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690247 GSM4690247 GEO Accession GSM4690247 SRX8814332 GSM4690248 SRP273093 PRJNA647773 SRS7078170 GSM4690248 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690248 GSM4690248 GEO Accession GSM4690248 SRX8814333 GSM4690249 SRP273093 PRJNA647773 SRS7078171 GSM4690249 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690249 GSM4690249 GEO Accession GSM4690249 SRX8814334 GSM4690250 SRP273093 PRJNA647773 SRS7078172 GSM4690250 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690250 GSM4690250 GEO Accession GSM4690250 SRX8814335 GSM4690251 SRP273093 PRJNA647773 SRS7078173 GSM4690251 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690251 GSM4690251 GEO Accession GSM4690251 SRX8814336 GSM4690252 SRP273093 PRJNA647773 SRS7078175 GSM4690252 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690252 GSM4690252 GEO Accession GSM4690252 SRX8814337 GSM4690253 SRP273093 PRJNA647773 SRS7078176 GSM4690253 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690253 GSM4690253 GEO Accession GSM4690253 SRX8814338 GSM4690254 SRP273093 PRJNA647773 SRS7078178 GSM4690254 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690254 GSM4690254 GEO Accession GSM4690254 SRX8814339 GSM4690255 SRP273093 PRJNA647773 SRS7078177 GSM4690255 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690255 GSM4690255 GEO Accession GSM4690255 SRX8814340 GSM4690256 SRP273093 PRJNA647773 SRS7078179 GSM4690256 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690256 GSM4690256 GEO Accession GSM4690256 SRX8814341 GSM4690257 SRP273093 PRJNA647773 SRS7078181 GSM4690257 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690257 GSM4690257 GEO Accession GSM4690257 SRX8814342 GSM4690258 SRP273093 PRJNA647773 SRS7078180 GSM4690258 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690258 GSM4690258 GEO Accession GSM4690258 SRX8814343 GSM4690259 SRP273093 PRJNA647773 SRS7078185 GSM4690259 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690259 GSM4690259 GEO Accession GSM4690259 SRX8814344 GSM4690260 SRP273093 PRJNA647773 SRS7078182 GSM4690260 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690260 GSM4690260 GEO Accession GSM4690260 SRX8814345 GSM4690261 SRP273093 PRJNA647773 SRS7078183 GSM4690261 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690261 GSM4690261 GEO Accession GSM4690261 SRX8814346 GSM4690262 SRP273093 PRJNA647773 SRS7078184 GSM4690262 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690262 GSM4690262 GEO Accession GSM4690262 SRX8814347 GSM4690263 SRP273093 PRJNA647773 SRS7078186 GSM4690263 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690263 GSM4690263 GEO Accession GSM4690263 SRX8814348 GSM4690264 SRP273093 PRJNA647773 SRS7078187 GSM4690264 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690264 GSM4690264 GEO Accession GSM4690264 SRX8814349 GSM4690265 SRP273093 PRJNA647773 SRS7078189 GSM4690265 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690265 GSM4690265 GEO Accession GSM4690265 SRX8814350 GSM4690266 SRP273093 PRJNA647773 SRS7078188 GSM4690266 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690266 GSM4690266 GEO Accession GSM4690266 SRX8814351 GSM4690267 SRP273093 PRJNA647773 SRS7078190 GSM4690267 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690267 GSM4690267 GEO Accession GSM4690267 SRX8814352 GSM4690268 SRP273093 PRJNA647773 SRS7078192 GSM4690268 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690268 GSM4690268 GEO Accession GSM4690268 SRX8814353 GSM4690269 SRP273093 PRJNA647773 SRS7078191 GSM4690269 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690269 GSM4690269 GEO Accession GSM4690269 SRX8814354 GSM4690270 SRP273093 PRJNA647773 SRS7078193 GSM4690270 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690270 GSM4690270 GEO Accession GSM4690270 SRX8814355 GSM4690271 SRP273093 PRJNA647773 SRS7078194 GSM4690271 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690271 GSM4690271 GEO Accession GSM4690271 SRX8814356 GSM4690272 SRP273093 PRJNA647773 SRS7078195 GSM4690272 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690272 GSM4690272 GEO Accession GSM4690272 SRX8814357 GSM4690273 SRP273093 PRJNA647773 SRS7078196 GSM4690273 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690273 GSM4690273 GEO Accession GSM4690273 SRX8814358 GSM4690274 SRP273093 PRJNA647773 SRS7078197 GSM4690274 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690274 GSM4690274 GEO Accession GSM4690274 SRX8814359 GSM4690275 SRP273093 PRJNA647773 SRS7078199 GSM4690275 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690275 GSM4690275 GEO Accession GSM4690275 SRX8814360 GSM4690276 SRP273093 PRJNA647773 SRS7078198 GSM4690276 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690276 GSM4690276 GEO Accession GSM4690276 SRX8814361 GSM4690277 SRP273093 PRJNA647773 SRS7078200 GSM4690277 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690277 GSM4690277 GEO Accession GSM4690277 SRX8814362 GSM4690278 SRP273093 PRJNA647773 SRS7078201 GSM4690278 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690278 GSM4690278 GEO Accession GSM4690278 SRX8814363 GSM4690279 SRP273093 PRJNA647773 SRS7078203 GSM4690279 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690279 GSM4690279 GEO Accession GSM4690279 SRX8814364 GSM4690280 SRP273093 PRJNA647773 SRS7078204 GSM4690280 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690280 GSM4690280 GEO Accession GSM4690280 SRX8814365 GSM4690281 SRP273093 PRJNA647773 SRS7078202 GSM4690281 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690281 GSM4690281 GEO Accession GSM4690281 SRX8814366 GSM4690282 SRP273093 PRJNA647773 SRS7078205 GSM4690282 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690282 GSM4690282 GEO Accession GSM4690282 SRX8814367 GSM4690283 SRP273093 PRJNA647773 SRS7078207 GSM4690283 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690283 GSM4690283 GEO Accession GSM4690283 SRX8814368 GSM4690284 SRP273093 PRJNA647773 SRS7078206 GSM4690284 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690284 GSM4690284 GEO Accession GSM4690284 SRX8814369 GSM4690285 SRP273093 PRJNA647773 SRS7078211 GSM4690285 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690285 GSM4690285 GEO Accession GSM4690285 SRX8814370 GSM4690286 SRP273093 PRJNA647773 SRS7078208 GSM4690286 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690286 GSM4690286 GEO Accession GSM4690286 SRX8814371 GSM4690287 SRP273093 PRJNA647773 SRS7078209 GSM4690287 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690287 GSM4690287 GEO Accession GSM4690287 SRX8814372 GSM4690288 SRP273093 PRJNA647773 SRS7078210 GSM4690288 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690288 GSM4690288 GEO Accession GSM4690288 SRX8814373 GSM4690289 SRP273093 PRJNA647773 SRS7078213 GSM4690289 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690289 GSM4690289 GEO Accession GSM4690289 SRX8814374 GSM4690290 SRP273093 PRJNA647773 SRS7078212 GSM4690290 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690290 GSM4690290 GEO Accession GSM4690290 SRX8814375 GSM4690291 SRP273093 PRJNA647773 SRS7078214 GSM4690291 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690291 GSM4690291 GEO Accession GSM4690291 SRX8814376 GSM4690292 SRP273093 PRJNA647773 SRS7078215 GSM4690292 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690292 GSM4690292 GEO Accession GSM4690292 SRX8814377 GSM4690293 SRP273093 PRJNA647773 SRS7078217 GSM4690293 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690293 GSM4690293 GEO Accession GSM4690293 SRX8814378 GSM4690294 SRP273093 PRJNA647773 SRS7078218 GSM4690294 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690294 GSM4690294 GEO Accession GSM4690294 SRX8814379 GSM4690295 SRP273093 PRJNA647773 SRS7078216 GSM4690295 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690295 GSM4690295 GEO Accession GSM4690295 SRX8814380 GSM4690296 SRP273093 PRJNA647773 SRS7078219 GSM4690296 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690296 GSM4690296 GEO Accession GSM4690296 SRX8814381 GSM4690297 SRP273093 PRJNA647773 SRS7078220 GSM4690297 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690297 GSM4690297 GEO Accession GSM4690297 SRX8814382 GSM4690298 SRP273093 PRJNA647773 SRS7078221 GSM4690298 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690298 GSM4690298 GEO Accession GSM4690298 SRX8814383 GSM4690299 SRP273093 PRJNA647773 SRS7078223 GSM4690299 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690299 GSM4690299 GEO Accession GSM4690299 SRX8814384 GSM4690300 SRP273093 PRJNA647773 SRS7078222 GSM4690300 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690300 GSM4690300 GEO Accession GSM4690300 SRX8814385 GSM4690301 SRP273093 PRJNA647773 SRS7078224 GSM4690301 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690301 GSM4690301 GEO Accession GSM4690301 SRX8814386 GSM4690302 SRP273093 PRJNA647773 SRS7078225 GSM4690302 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690302 GSM4690302 GEO Accession GSM4690302 SRX8814387 GSM4690303 SRP273093 PRJNA647773 SRS7078227 GSM4690303 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690303 GSM4690303 GEO Accession GSM4690303 SRX8814388 GSM4690304 SRP273093 PRJNA647773 SRS7078226 GSM4690304 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690304 GSM4690304 GEO Accession GSM4690304 SRX8814389 GSM4690305 SRP273093 PRJNA647773 SRS7078229 GSM4690305 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690305 GSM4690305 GEO Accession GSM4690305 SRX8814390 GSM4690306 SRP273093 PRJNA647773 SRS7078228 GSM4690306 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690306 GSM4690306 GEO Accession GSM4690306 SRX8814391 GSM4690307 SRP273093 PRJNA647773 SRS7078230 GSM4690307 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690307 GSM4690307 GEO Accession GSM4690307 SRX8814392 GSM4690308 SRP273093 PRJNA647773 SRS7078231 GSM4690308 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690308 GSM4690308 GEO Accession GSM4690308 SRX8814393 GSM4690309 SRP273093 PRJNA647773 SRS7078232 GSM4690309 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690309 GSM4690309 GEO Accession GSM4690309 SRX8814394 GSM4690310 SRP273093 PRJNA647773 SRS7078233 GSM4690310 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690310 GSM4690310 GEO Accession GSM4690310 SRX8814395 GSM4690311 SRP273093 PRJNA647773 SRS7078234 GSM4690311 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690311 GSM4690311 GEO Accession GSM4690311 SRX8814396 GSM4690312 SRP273093 PRJNA647773 SRS7078235 GSM4690312 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690312 GSM4690312 GEO Accession GSM4690312 SRX8814397 GSM4690313 SRP273093 PRJNA647773 SRS7078236 GSM4690313 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690313 GSM4690313 GEO Accession GSM4690313 SRX8814398 GSM4690314 SRP273093 PRJNA647773 SRS7078237 GSM4690314 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690314 GSM4690314 GEO Accession GSM4690314 SRX8814399 GSM4690315 SRP273093 PRJNA647773 SRS7078238 GSM4690315 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690315 GSM4690315 GEO Accession GSM4690315 SRX8814400 GSM4690316 SRP273093 PRJNA647773 SRS7078240 GSM4690316 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690316 GSM4690316 GEO Accession GSM4690316 SRX8814401 GSM4690317 SRP273093 PRJNA647773 SRS7078239 GSM4690317 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690317 GSM4690317 GEO Accession GSM4690317 SRX8814402 GSM4690318 SRP273093 PRJNA647773 SRS7078241 GSM4690318 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690318 GSM4690318 GEO Accession GSM4690318 SRX8814403 GSM4690319 SRP273093 PRJNA647773 SRS7078242 GSM4690319 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690319 GSM4690319 GEO Accession GSM4690319 SRX8814404 GSM4690320 SRP273093 PRJNA647773 SRS7078243 GSM4690320 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690320 GSM4690320 GEO Accession GSM4690320 SRX8814405 GSM4690321 SRP273093 PRJNA647773 SRS7078244 GSM4690321 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690321 GSM4690321 GEO Accession GSM4690321 SRX8814406 GSM4690322 SRP273093 PRJNA647773 SRS7078246 GSM4690322 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690322 GSM4690322 GEO Accession GSM4690322 SRX8814407 GSM4690323 SRP273093 PRJNA647773 SRS7078245 GSM4690323 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690323 GSM4690323 GEO Accession GSM4690323 SRX8814408 GSM4690324 SRP273093 PRJNA647773 SRS7078247 GSM4690324 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690324 GSM4690324 GEO Accession GSM4690324 SRX8814409 GSM4690325 SRP273093 PRJNA647773 SRS7078250 GSM4690325 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690325 GSM4690325 GEO Accession GSM4690325 SRX8814410 GSM4690326 SRP273093 PRJNA647773 SRS7078248 GSM4690326 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690326 GSM4690326 GEO Accession GSM4690326 SRX8814411 GSM4690327 SRP273093 PRJNA647773 SRS7078249 GSM4690327 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690327 GSM4690327 GEO Accession GSM4690327 SRX8814412 GSM4690328 SRP273093 PRJNA647773 SRS7078251 GSM4690328 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690328 GSM4690328 GEO Accession GSM4690328 SRX8814413 GSM4690329 SRP273093 PRJNA647773 SRS7078252 GSM4690329 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690329 GSM4690329 GEO Accession GSM4690329 SRX8814414 GSM4690330 SRP273093 PRJNA647773 SRS7078254 GSM4690330 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690330 GSM4690330 GEO Accession GSM4690330 SRX8814415 GSM4690331 SRP273093 PRJNA647773 SRS7078253 GSM4690331 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690331 GSM4690331 GEO Accession GSM4690331 SRX8814416 GSM4690332 SRP273093 PRJNA647773 SRS7078255 GSM4690332 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690332 GSM4690332 GEO Accession GSM4690332 SRX8814417 GSM4690333 SRP273093 PRJNA647773 SRS7078256 GSM4690333 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690333 GSM4690333 GEO Accession GSM4690333 SRX8814418 GSM4690334 SRP273093 PRJNA647773 SRS7078257 GSM4690334 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690334 GSM4690334 GEO Accession GSM4690334 SRX8814419 GSM4690335 SRP273093 PRJNA647773 SRS7078258 GSM4690335 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690335 GSM4690335 GEO Accession GSM4690335 SRX8814420 GSM4690336 SRP273093 PRJNA647773 SRS7078259 GSM4690336 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690336 GSM4690336 GEO Accession GSM4690336 SRX8814421 GSM4690337 SRP273093 PRJNA647773 SRS7078260 GSM4690337 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690337 GSM4690337 GEO Accession GSM4690337 SRX8814422 GSM4690338 SRP273093 PRJNA647773 SRS7078262 GSM4690338 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690338 GSM4690338 GEO Accession GSM4690338 SRX8814423 GSM4690339 SRP273093 PRJNA647773 SRS7078261 GSM4690339 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690339 GSM4690339 GEO Accession GSM4690339 SRX8814424 GSM4690340 SRP273093 PRJNA647773 SRS7078263 GSM4690340 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690340 GSM4690340 GEO Accession GSM4690340 SRX8814425 GSM4690341 SRP273093 PRJNA647773 SRS7078266 GSM4690341 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690341 GSM4690341 GEO Accession GSM4690341 SRX8814426 GSM4690342 SRP273093 PRJNA647773 SRS7078265 GSM4690342 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690342 GSM4690342 GEO Accession GSM4690342 SRX8814427 GSM4690343 SRP273093 PRJNA647773 SRS7078267 GSM4690343 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690343 GSM4690343 GEO Accession GSM4690343 SRX8814428 GSM4690344 SRP273093 PRJNA647773 SRS7078264 GSM4690344 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690344 GSM4690344 GEO Accession GSM4690344 SRX8814429 GSM4690345 SRP273093 PRJNA647773 SRS7078268 GSM4690345 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690345 GSM4690345 GEO Accession GSM4690345 SRX8814430 GSM4690346 SRP273093 PRJNA647773 SRS7078269 GSM4690346 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690346 GSM4690346 GEO Accession GSM4690346 SRX8814431 GSM4690347 SRP273093 PRJNA647773 SRS7078270 GSM4690347 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690347 GSM4690347 GEO Accession GSM4690347 SRX8814432 GSM4690348 SRP273093 PRJNA647773 SRS7078271 GSM4690348 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690348 GSM4690348 GEO Accession GSM4690348 SRX8814433 GSM4690349 SRP273093 PRJNA647773 SRS7078273 GSM4690349 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690349 GSM4690349 GEO Accession GSM4690349 SRX8814434 GSM4690350 SRP273093 PRJNA647773 SRS7078272 GSM4690350 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690350 GSM4690350 GEO Accession GSM4690350 SRX8814435 GSM4690351 SRP273093 PRJNA647773 SRS7078274 GSM4690351 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690351 GSM4690351 GEO Accession GSM4690351 SRX8814436 GSM4690352 SRP273093 PRJNA647773 SRS7078275 GSM4690352 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690352 GSM4690352 GEO Accession GSM4690352 SRX8814437 GSM4690353 SRP273093 PRJNA647773 SRS7078277 GSM4690353 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690353 GSM4690353 GEO Accession GSM4690353 SRX8814438 GSM4690354 SRP273093 PRJNA647773 SRS7078276 GSM4690354 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690354 GSM4690354 GEO Accession GSM4690354 SRX8814439 GSM4690355 SRP273093 PRJNA647773 SRS7076748 GSM4690355 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690355 GSM4690355 GEO Accession GSM4690355 SRX8814440 GSM4690356 SRP273093 PRJNA647773 SRS7076749 GSM4690356 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690356 GSM4690356 GEO Accession GSM4690356 SRX8814441 GSM4690357 SRP273093 PRJNA647773 SRS7076750 GSM4690357 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690357 GSM4690357 GEO Accession GSM4690357 SRX8814442 GSM4690358 SRP273093 PRJNA647773 SRS7076089 GSM4690358 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690358 GSM4690358 GEO Accession GSM4690358 SRX8814443 GSM4690359 SRP273093 PRJNA647773 SRS7076090 GSM4690359 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690359 GSM4690359 GEO Accession GSM4690359 SRX8814444 GSM4690360 SRP273093 PRJNA647773 SRS7076751 GSM4690360 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690360 GSM4690360 GEO Accession GSM4690360 SRX8814445 GSM4690361 SRP273093 PRJNA647773 SRS7076752 GSM4690361 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690361 GSM4690361 GEO Accession GSM4690361 SRX8814446 GSM4690362 SRP273093 PRJNA647773 SRS7076091 GSM4690362 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690362 GSM4690362 GEO Accession GSM4690362 SRX8814447 GSM4690363 SRP273093 PRJNA647773 SRS7076753 GSM4690363 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690363 GSM4690363 GEO Accession GSM4690363 SRX8814448 GSM4690364 SRP273093 PRJNA647773 SRS7076755 GSM4690364 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690364 GSM4690364 GEO Accession GSM4690364 SRX8814449 GSM4690365 SRP273093 PRJNA647773 SRS7076754 GSM4690365 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690365 GSM4690365 GEO Accession GSM4690365 SRX8814450 GSM4690366 SRP273093 PRJNA647773 SRS7076756 GSM4690366 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690366 GSM4690366 GEO Accession GSM4690366 SRX8814451 GSM4690367 SRP273093 PRJNA647773 SRS7076757 GSM4690367 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690367 GSM4690367 GEO Accession GSM4690367 SRX8814452 GSM4690368 SRP273093 PRJNA647773 SRS7076758 GSM4690368 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690368 GSM4690368 GEO Accession GSM4690368 SRX8814453 GSM4690369 SRP273093 PRJNA647773 SRS7076759 GSM4690369 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690369 GSM4690369 GEO Accession GSM4690369 SRX8814454 GSM4690370 SRP273093 PRJNA647773 SRS7076760 GSM4690370 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690370 GSM4690370 GEO Accession GSM4690370 SRX8814455 GSM4690371 SRP273093 PRJNA647773 SRS7076761 GSM4690371 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690371 GSM4690371 GEO Accession GSM4690371 SRX8814456 GSM4690372 SRP273093 PRJNA647773 SRS7076762 GSM4690372 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690372 GSM4690372 GEO Accession GSM4690372 SRX8814457 GSM4690373 SRP273093 PRJNA647773 SRS7076763 GSM4690373 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690373 GSM4690373 GEO Accession GSM4690373 SRX8814458 GSM4690374 SRP273093 PRJNA647773 SRS7076764 GSM4690374 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690374 GSM4690374 GEO Accession GSM4690374 SRX8814459 GSM4690375 SRP273093 PRJNA647773 SRS7076766 GSM4690375 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690375 GSM4690375 GEO Accession GSM4690375 SRX8814460 GSM4690376 SRP273093 PRJNA647773 SRS7076765 GSM4690376 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690376 GSM4690376 GEO Accession GSM4690376 SRX8814461 GSM4690377 SRP273093 PRJNA647773 SRS7076767 GSM4690377 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690377 GSM4690377 GEO Accession GSM4690377 SRX8814462 GSM4690378 SRP273093 PRJNA647773 SRS7076768 GSM4690378 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690378 GSM4690378 GEO Accession GSM4690378 SRX8814463 GSM4690379 SRP273093 PRJNA647773 SRS7076769 GSM4690379 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690379 GSM4690379 GEO Accession GSM4690379 SRX8814464 GSM4690380 SRP273093 PRJNA647773 SRS7076770 GSM4690380 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690380 GSM4690380 GEO Accession GSM4690380 SRX8814465 GSM4690381 SRP273093 PRJNA647773 SRS7076771 GSM4690381 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690381 GSM4690381 GEO Accession GSM4690381 SRX8814466 GSM4690382 SRP273093 PRJNA647773 SRS7076772 GSM4690382 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690382 GSM4690382 GEO Accession GSM4690382 SRX8814467 GSM4690383 SRP273093 PRJNA647773 SRS7076773 GSM4690383 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690383 GSM4690383 GEO Accession GSM4690383 SRX8814468 GSM4690384 SRP273093 PRJNA647773 SRS7076774 GSM4690384 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690384 GSM4690384 GEO Accession GSM4690384 SRX8814469 GSM4690385 SRP273093 PRJNA647773 SRS7076777 GSM4690385 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690385 GSM4690385 GEO Accession GSM4690385 SRX8814470 GSM4690386 SRP273093 PRJNA647773 SRS7076775 GSM4690386 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690386 GSM4690386 GEO Accession GSM4690386 SRX8814471 GSM4690387 SRP273093 PRJNA647773 SRS7076776 GSM4690387 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690387 GSM4690387 GEO Accession GSM4690387 SRX8814472 GSM4690388 SRP273093 PRJNA647773 SRS7076778 GSM4690388 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690388 GSM4690388 GEO Accession GSM4690388 SRX8814473 GSM4690389 SRP273093 PRJNA647773 SRS7076779 GSM4690389 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690389 GSM4690389 GEO Accession GSM4690389 SRX8814474 GSM4690390 SRP273093 PRJNA647773 SRS7076780 GSM4690390 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690390 GSM4690390 GEO Accession GSM4690390 SRX8814475 GSM4690391 SRP273093 PRJNA647773 SRS7076781 GSM4690391 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690391 GSM4690391 GEO Accession GSM4690391 SRX8814476 GSM4690392 SRP273093 PRJNA647773 SRS7076782 GSM4690392 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690392 GSM4690392 GEO Accession GSM4690392 SRX8814477 GSM4690393 SRP273093 PRJNA647773 SRS7076783 GSM4690393 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690393 GSM4690393 GEO Accession GSM4690393 SRX8814478 GSM4690394 SRP273093 PRJNA647773 SRS7076784 GSM4690394 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690394 GSM4690394 GEO Accession GSM4690394 SRX8814479 GSM4690395 SRP273093 PRJNA647773 SRS7076785 GSM4690395 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690395 GSM4690395 GEO Accession GSM4690395 SRX8814480 GSM4690396 SRP273093 PRJNA647773 SRS7076786 GSM4690396 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690396 GSM4690396 GEO Accession GSM4690396 SRX8814481 GSM4690397 SRP273093 PRJNA647773 SRS7076787 GSM4690397 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690397 GSM4690397 GEO Accession GSM4690397 SRX8814482 GSM4690398 SRP273093 PRJNA647773 SRS7076790 GSM4690398 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690398 GSM4690398 GEO Accession GSM4690398 SRX8814483 GSM4690399 SRP273093 PRJNA647773 SRS7076788 GSM4690399 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690399 GSM4690399 GEO Accession GSM4690399 SRX8814484 GSM4690400 SRP273093 PRJNA647773 SRS7076789 GSM4690400 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690400 GSM4690400 GEO Accession GSM4690400 SRX8814485 GSM4690401 SRP273093 PRJNA647773 SRS7076791 GSM4690401 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690401 GSM4690401 GEO Accession GSM4690401 SRX8814486 GSM4690402 SRP273093 PRJNA647773 SRS7076793 GSM4690402 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690402 GSM4690402 GEO Accession GSM4690402 SRX8814487 GSM4690403 SRP273093 PRJNA647773 SRS7076792 GSM4690403 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690403 GSM4690403 GEO Accession GSM4690403 SRX8814488 GSM4690404 SRP273093 PRJNA647773 SRS7076794 GSM4690404 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690404 GSM4690404 GEO Accession GSM4690404 SRX8814489 GSM4690405 SRP273093 PRJNA647773 SRS7076795 GSM4690405 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690405 GSM4690405 GEO Accession GSM4690405 SRX8814490 GSM4690406 SRP273093 PRJNA647773 SRS7076797 GSM4690406 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690406 GSM4690406 GEO Accession GSM4690406 SRX8814491 GSM4690407 SRP273093 PRJNA647773 SRS7076796 GSM4690407 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690407 GSM4690407 GEO Accession GSM4690407 SRX8814492 GSM4690408 SRP273093 PRJNA647773 SRS7076799 GSM4690408 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690408 GSM4690408 GEO Accession GSM4690408 SRX8814493 GSM4690409 SRP273093 PRJNA647773 SRS7076798 GSM4690409 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690409 GSM4690409 GEO Accession GSM4690409 SRX8814494 GSM4690410 SRP273093 PRJNA647773 SRS7076800 GSM4690410 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690410 GSM4690410 GEO Accession GSM4690410 SRX8814495 GSM4690411 SRP273093 PRJNA647773 SRS7076801 GSM4690411 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690411 GSM4690411 GEO Accession GSM4690411 SRX8814496 GSM4690412 SRP273093 PRJNA647773 SRS7076802 GSM4690412 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690412 GSM4690412 GEO Accession GSM4690412 SRX8814497 GSM4690413 SRP273093 PRJNA647773 SRS7076803 GSM4690413 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690413 GSM4690413 GEO Accession GSM4690413 SRX8814498 GSM4690414 SRP273093 PRJNA647773 SRS7076804 GSM4690414 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690414 GSM4690414 GEO Accession GSM4690414 SRX8814499 GSM4690415 SRP273093 PRJNA647773 SRS7076805 GSM4690415 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690415 GSM4690415 GEO Accession GSM4690415 SRX8814500 GSM4690416 SRP273093 PRJNA647773 SRS7076806 GSM4690416 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690416 GSM4690416 GEO Accession GSM4690416 SRX8814501 GSM4690417 SRP273093 PRJNA647773 SRS7076807 GSM4690417 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690417 GSM4690417 GEO Accession GSM4690417 SRX8814502 GSM4690418 SRP273093 PRJNA647773 SRS7076808 GSM4690418 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690418 GSM4690418 GEO Accession GSM4690418 SRX8814503 GSM4690419 SRP273093 PRJNA647773 SRS7076809 GSM4690419 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690419 GSM4690419 GEO Accession GSM4690419 SRX8814504 GSM4690420 SRP273093 PRJNA647773 SRS7076810 GSM4690420 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690420 GSM4690420 GEO Accession GSM4690420 SRX8814505 GSM4690421 SRP273093 PRJNA647773 SRS7076811 GSM4690421 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690421 GSM4690421 GEO Accession GSM4690421 SRX8814506 GSM4690422 SRP273093 PRJNA647773 SRS7076812 GSM4690422 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690422 GSM4690422 GEO Accession GSM4690422 SRX8814507 GSM4690423 SRP273093 PRJNA647773 SRS7076813 GSM4690423 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690423 GSM4690423 GEO Accession GSM4690423 SRX8814508 GSM4690424 SRP273093 PRJNA647773 SRS7076814 GSM4690424 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690424 GSM4690424 GEO Accession GSM4690424 SRX8814509 GSM4690425 SRP273093 PRJNA647773 SRS7076815 GSM4690425 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690425 GSM4690425 GEO Accession GSM4690425 SRX8814510 GSM4690426 SRP273093 PRJNA647773 SRS7076816 GSM4690426 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690426 GSM4690426 GEO Accession GSM4690426 SRX8814511 GSM4690427 SRP273093 PRJNA647773 SRS7076817 GSM4690427 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690427 GSM4690427 GEO Accession GSM4690427 SRX8814512 GSM4690428 SRP273093 PRJNA647773 SRS7076818 GSM4690428 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690428 GSM4690428 GEO Accession GSM4690428 SRX8814513 GSM4690429 SRP273093 PRJNA647773 SRS7076820 GSM4690429 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690429 GSM4690429 GEO Accession GSM4690429 SRX8814514 GSM4690430 SRP273093 PRJNA647773 SRS7076819 GSM4690430 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690430 GSM4690430 GEO Accession GSM4690430 SRX8814515 GSM4690431 SRP273093 PRJNA647773 SRS7076821 GSM4690431 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690431 GSM4690431 GEO Accession GSM4690431 SRX8814516 GSM4690432 SRP273093 PRJNA647773 SRS7076822 GSM4690432 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690432 GSM4690432 GEO Accession GSM4690432 SRX8814517 GSM4690433 SRP273093 PRJNA647773 SRS7076823 GSM4690433 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690433 GSM4690433 GEO Accession GSM4690433 SRX8814518 GSM4690434 SRP273093 PRJNA647773 SRS7076824 GSM4690434 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690434 GSM4690434 GEO Accession GSM4690434 SRX8814519 GSM4690435 SRP273093 PRJNA647773 SRS7076827 GSM4690435 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690435 GSM4690435 GEO Accession GSM4690435 SRX8814520 GSM4690436 SRP273093 PRJNA647773 SRS7076826 GSM4690436 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690436 GSM4690436 GEO Accession GSM4690436 SRX8814521 GSM4690437 SRP273093 PRJNA647773 SRS7076825 GSM4690437 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690437 GSM4690437 GEO Accession GSM4690437 SRX8814522 GSM4690438 SRP273093 PRJNA647773 SRS7076828 GSM4690438 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690438 GSM4690438 GEO Accession GSM4690438 SRX8814523 GSM4690439 SRP273093 PRJNA647773 SRS7076829 GSM4690439 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690439 GSM4690439 GEO Accession GSM4690439 SRX8814524 GSM4690440 SRP273093 PRJNA647773 SRS7076830 GSM4690440 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690440 GSM4690440 GEO Accession GSM4690440 SRX8814525 GSM4690441 SRP273093 PRJNA647773 SRS7076831 GSM4690441 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690441 GSM4690441 GEO Accession GSM4690441 SRX8814526 GSM4690442 SRP273093 PRJNA647773 SRS7076832 GSM4690442 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690442 GSM4690442 GEO Accession GSM4690442 SRX8814527 GSM4690443 SRP273093 PRJNA647773 SRS7076833 GSM4690443 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690443 GSM4690443 GEO Accession GSM4690443 SRX8814528 GSM4690444 SRP273093 PRJNA647773 SRS7076835 GSM4690444 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690444 GSM4690444 GEO Accession GSM4690444 SRX8814529 GSM4690445 SRP273093 PRJNA647773 SRS7076834 GSM4690445 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690445 GSM4690445 GEO Accession GSM4690445 SRX8814530 GSM4690446 SRP273093 PRJNA647773 SRS7076836 GSM4690446 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690446 GSM4690446 GEO Accession GSM4690446 SRX8814531 GSM4690447 SRP273093 PRJNA647773 SRS7076837 GSM4690447 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690447 GSM4690447 GEO Accession GSM4690447 SRX8814532 GSM4690448 SRP273093 PRJNA647773 SRS7076838 GSM4690448 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690448 GSM4690448 GEO Accession GSM4690448 SRX8814533 GSM4690449 SRP273093 PRJNA647773 SRS7076839 GSM4690449 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690449 GSM4690449 GEO Accession GSM4690449 SRX8814534 GSM4690450 SRP273093 PRJNA647773 SRS7076841 GSM4690450 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690450 GSM4690450 GEO Accession GSM4690450 SRX8814535 GSM4690451 SRP273093 PRJNA647773 SRS7076840 GSM4690451 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690451 GSM4690451 GEO Accession GSM4690451 SRX8814536 GSM4690452 SRP273093 PRJNA647773 SRS7076842 GSM4690452 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690452 GSM4690452 GEO Accession GSM4690452 SRX8814537 GSM4690453 SRP273093 PRJNA647773 SRS7076843 GSM4690453 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690453 GSM4690453 GEO Accession GSM4690453 SRX8814538 GSM4690454 SRP273093 PRJNA647773 SRS7076844 GSM4690454 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690454 GSM4690454 GEO Accession GSM4690454 SRX8814539 GSM4690455 SRP273093 PRJNA647773 SRS7076845 GSM4690455 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690455 GSM4690455 GEO Accession GSM4690455 SRX8814540 GSM4690456 SRP273093 PRJNA647773 SRS7076846 GSM4690456 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690456 GSM4690456 GEO Accession GSM4690456 SRX8814541 GSM4690457 SRP273093 PRJNA647773 SRS7076847 GSM4690457 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690457 GSM4690457 GEO Accession GSM4690457 SRX8814542 GSM4690458 SRP273093 PRJNA647773 SRS7076849 GSM4690458 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690458 GSM4690458 GEO Accession GSM4690458 SRX8814543 GSM4690459 SRP273093 PRJNA647773 SRS7076848 GSM4690459 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690459 GSM4690459 GEO Accession GSM4690459 SRX8814544 GSM4690460 SRP273093 PRJNA647773 SRS7076851 GSM4690460 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690460 GSM4690460 GEO Accession GSM4690460 SRX8814545 GSM4690461 SRP273093 PRJNA647773 SRS7076850 GSM4690461 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690461 GSM4690461 GEO Accession GSM4690461 SRX8814546 GSM4690462 SRP273093 PRJNA647773 SRS7076852 GSM4690462 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690462 GSM4690462 GEO Accession GSM4690462 SRX8814547 GSM4690463 SRP273093 PRJNA647773 SRS7076853 GSM4690463 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690463 GSM4690463 GEO Accession GSM4690463 SRX8814548 GSM4690464 SRP273093 PRJNA647773 SRS7076854 GSM4690464 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690464 GSM4690464 GEO Accession GSM4690464 SRX8814549 GSM4690465 SRP273093 PRJNA647773 SRS7076856 GSM4690465 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690465 GSM4690465 GEO Accession GSM4690465 SRX8814550 GSM4690466 SRP273093 PRJNA647773 SRS7076855 GSM4690466 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690466 GSM4690466 GEO Accession GSM4690466 SRX8814551 GSM4690467 SRP273093 PRJNA647773 SRS7076857 GSM4690467 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690467 GSM4690467 GEO Accession GSM4690467 SRX8814552 GSM4690468 SRP273093 PRJNA647773 SRS7076858 GSM4690468 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690468 GSM4690468 GEO Accession GSM4690468 SRX8814553 GSM4690469 SRP273093 PRJNA647773 SRS7076859 GSM4690469 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690469 GSM4690469 GEO Accession GSM4690469 SRX8814554 GSM4690470 SRP273093 PRJNA647773 SRS7076860 GSM4690470 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690470 GSM4690470 GEO Accession GSM4690470 SRX8814555 GSM4690471 SRP273093 PRJNA647773 SRS7076861 GSM4690471 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690471 GSM4690471 GEO Accession GSM4690471 SRX8814556 GSM4690472 SRP273093 PRJNA647773 SRS7076862 GSM4690472 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690472 GSM4690472 GEO Accession GSM4690472 SRX8814557 GSM4690473 SRP273093 PRJNA647773 SRS7076863 GSM4690473 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690473 GSM4690473 GEO Accession GSM4690473 SRX8814558 GSM4690474 SRP273093 PRJNA647773 SRS7076864 GSM4690474 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690474 GSM4690474 GEO Accession GSM4690474 SRX8814559 GSM4690475 SRP273093 PRJNA647773 SRS7076865 GSM4690475 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690475 GSM4690475 GEO Accession GSM4690475 SRX8814560 GSM4690476 SRP273093 PRJNA647773 SRS7076866 GSM4690476 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690476 GSM4690476 GEO Accession GSM4690476 SRX8814561 GSM4690477 SRP273093 PRJNA647773 SRS7076867 GSM4690477 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690477 GSM4690477 GEO Accession GSM4690477 SRX8814562 GSM4690478 SRP273093 PRJNA647773 SRS7076868 GSM4690478 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690478 GSM4690478 GEO Accession GSM4690478 SRX8814563 GSM4690479 SRP273093 PRJNA647773 SRS7076869 GSM4690479 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690479 GSM4690479 GEO Accession GSM4690479 SRX8814564 GSM4690480 SRP273093 PRJNA647773 SRS7076870 GSM4690480 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690480 GSM4690480 GEO Accession GSM4690480 SRX8814565 GSM4690481 SRP273093 PRJNA647773 SRS7076872 GSM4690481 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690481 GSM4690481 GEO Accession GSM4690481 SRX8814566 GSM4690482 SRP273093 PRJNA647773 SRS7076871 GSM4690482 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690482 GSM4690482 GEO Accession GSM4690482 SRX8814567 GSM4690483 SRP273093 PRJNA647773 SRS7076873 GSM4690483 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690483 GSM4690483 GEO Accession GSM4690483 SRX8814568 GSM4690484 SRP273093 PRJNA647773 SRS7076874 GSM4690484 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690484 GSM4690484 GEO Accession GSM4690484 SRX8814569 GSM4690485 SRP273093 PRJNA647773 SRS7076875 GSM4690485 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690485 GSM4690485 GEO Accession GSM4690485 SRX8814570 GSM4690486 SRP273093 PRJNA647773 SRS7076876 GSM4690486 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690486 GSM4690486 GEO Accession GSM4690486 SRX8814571 GSM4690487 SRP273093 PRJNA647773 SRS7076877 GSM4690487 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690487 GSM4690487 GEO Accession GSM4690487 SRX8814572 GSM4690488 SRP273093 PRJNA647773 SRS7076880 GSM4690488 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690488 GSM4690488 GEO Accession GSM4690488 SRX8814573 GSM4690489 SRP273093 PRJNA647773 SRS7076878 GSM4690489 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690489 GSM4690489 GEO Accession GSM4690489 SRX8814574 GSM4690490 SRP273093 PRJNA647773 SRS7076879 GSM4690490 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690490 GSM4690490 GEO Accession GSM4690490 SRX8814575 GSM4690491 SRP273093 PRJNA647773 SRS7076881 GSM4690491 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690491 GSM4690491 GEO Accession GSM4690491 SRX8814576 GSM4690492 SRP273093 PRJNA647773 SRS7076882 GSM4690492 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690492 GSM4690492 GEO Accession GSM4690492 SRX8814577 GSM4690493 SRP273093 PRJNA647773 SRS7076883 GSM4690493 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690493 GSM4690493 GEO Accession GSM4690493 SRX8814578 GSM4690494 SRP273093 PRJNA647773 SRS7076884 GSM4690494 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690494 GSM4690494 GEO Accession GSM4690494 SRX8814579 GSM4690495 SRP273093 PRJNA647773 SRS7076885 GSM4690495 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690495 GSM4690495 GEO Accession GSM4690495 SRX8814580 GSM4690496 SRP273093 PRJNA647773 SRS7076887 GSM4690496 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690496 GSM4690496 GEO Accession GSM4690496 SRX8814581 GSM4690497 SRP273093 PRJNA647773 SRS7076886 GSM4690497 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690497 GSM4690497 GEO Accession GSM4690497 SRX8814582 GSM4690498 SRP273093 PRJNA647773 SRS7076889 GSM4690498 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690498 GSM4690498 GEO Accession GSM4690498 SRX8814583 GSM4690499 SRP273093 PRJNA647773 SRS7076888 GSM4690499 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690499 GSM4690499 GEO Accession GSM4690499 SRX8814584 GSM4690500 SRP273093 PRJNA647773 SRS7076890 GSM4690500 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690500 GSM4690500 GEO Accession GSM4690500 SRX8814585 GSM4690501 SRP273093 PRJNA647773 SRS7076892 GSM4690501 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690501 GSM4690501 GEO Accession GSM4690501 SRX8814586 GSM4690502 SRP273093 PRJNA647773 SRS7076891 GSM4690502 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690502 GSM4690502 GEO Accession GSM4690502 SRX8814587 GSM4690503 SRP273093 PRJNA647773 SRS7076894 GSM4690503 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690503 GSM4690503 GEO Accession GSM4690503 SRX8814588 GSM4690504 SRP273093 PRJNA647773 SRS7076893 GSM4690504 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690504 GSM4690504 GEO Accession GSM4690504 SRX8814589 GSM4690505 SRP273093 PRJNA647773 SRS7076895 GSM4690505 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690505 GSM4690505 GEO Accession GSM4690505 SRX8814590 GSM4690506 SRP273093 PRJNA647773 SRS7076897 GSM4690506 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690506 GSM4690506 GEO Accession GSM4690506 SRX8814591 GSM4690507 SRP273093 PRJNA647773 SRS7076896 GSM4690507 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690507 GSM4690507 GEO Accession GSM4690507 SRX8814592 GSM4690508 SRP273093 PRJNA647773 SRS7076900 GSM4690508 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690508 GSM4690508 GEO Accession GSM4690508 SRX8814593 GSM4690509 SRP273093 PRJNA647773 SRS7076898 GSM4690509 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690509 GSM4690509 GEO Accession GSM4690509 SRX8814594 GSM4690510 SRP273093 PRJNA647773 SRS7076899 GSM4690510 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690510 GSM4690510 GEO Accession GSM4690510 SRX8814595 GSM4690511 SRP273093 PRJNA647773 SRS7076901 GSM4690511 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690511 GSM4690511 GEO Accession GSM4690511 SRX8814596 GSM4690512 SRP273093 PRJNA647773 SRS7076902 GSM4690512 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690512 GSM4690512 GEO Accession GSM4690512 SRX8814597 GSM4690513 SRP273093 PRJNA647773 SRS7076903 GSM4690513 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690513 GSM4690513 GEO Accession GSM4690513 SRX8814598 GSM4690514 SRP273093 PRJNA647773 SRS7076904 GSM4690514 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690514 GSM4690514 GEO Accession GSM4690514 SRX8814599 GSM4690515 SRP273093 PRJNA647773 SRS7076905 GSM4690515 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690515 GSM4690515 GEO Accession GSM4690515 SRX8814600 GSM4690516 SRP273093 PRJNA647773 SRS7076906 GSM4690516 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690516 GSM4690516 GEO Accession GSM4690516 SRX8814601 GSM4690517 SRP273093 PRJNA647773 SRS7076907 GSM4690517 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690517 GSM4690517 GEO Accession GSM4690517 SRX8814602 GSM4690518 SRP273093 PRJNA647773 SRS7076909 GSM4690518 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690518 GSM4690518 GEO Accession GSM4690518 SRX8814603 GSM4690519 SRP273093 PRJNA647773 SRS7076908 GSM4690519 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690519 GSM4690519 GEO Accession GSM4690519 SRX8814604 GSM4690520 SRP273093 PRJNA647773 SRS7076910 GSM4690520 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690520 GSM4690520 GEO Accession GSM4690520 SRX8814605 GSM4690521 SRP273093 PRJNA647773 SRS7076913 GSM4690521 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690521 GSM4690521 GEO Accession GSM4690521 SRX8814606 GSM4690522 SRP273093 PRJNA647773 SRS7076911 GSM4690522 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690522 GSM4690522 GEO Accession GSM4690522 SRX8814607 GSM4690523 SRP273093 PRJNA647773 SRS7076912 GSM4690523 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690523 GSM4690523 GEO Accession GSM4690523 SRX8814608 GSM4690524 SRP273093 PRJNA647773 SRS7076914 GSM4690524 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690524 GSM4690524 GEO Accession GSM4690524 SRX8814609 GSM4690525 SRP273093 PRJNA647773 SRS7076915 GSM4690525 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690525 GSM4690525 GEO Accession GSM4690525 SRX8814610 GSM4690526 SRP273093 PRJNA647773 SRS7076916 GSM4690526 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690526 GSM4690526 GEO Accession GSM4690526 SRX8814611 GSM4690527 SRP273093 PRJNA647773 SRS7076917 GSM4690527 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690527 GSM4690527 GEO Accession GSM4690527 SRX8814612 GSM4690528 SRP273093 PRJNA647773 SRS7076918 GSM4690528 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690528 GSM4690528 GEO Accession GSM4690528 SRX8814613 GSM4690529 SRP273093 PRJNA647773 SRS7076919 GSM4690529 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690529 GSM4690529 GEO Accession GSM4690529 SRX8814614 GSM4690530 SRP273093 PRJNA647773 SRS7076920 GSM4690530 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690530 GSM4690530 GEO Accession GSM4690530 SRX8814615 GSM4690531 SRP273093 PRJNA647773 SRS7076921 GSM4690531 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690531 GSM4690531 GEO Accession GSM4690531 SRX8814616 GSM4690532 SRP273093 PRJNA647773 SRS7076922 GSM4690532 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690532 GSM4690532 GEO Accession GSM4690532 SRX8814617 GSM4690533 SRP273093 PRJNA647773 SRS7076923 GSM4690533 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690533 GSM4690533 GEO Accession GSM4690533 SRX8814618 GSM4690534 SRP273093 PRJNA647773 SRS7076924 GSM4690534 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690534 GSM4690534 GEO Accession GSM4690534 SRX8814619 GSM4690535 SRP273093 PRJNA647773 SRS7076925 GSM4690535 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690535 GSM4690535 GEO Accession GSM4690535 SRX8814620 GSM4690536 SRP273093 PRJNA647773 SRS7076926 GSM4690536 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690536 GSM4690536 GEO Accession GSM4690536 SRX8814621 GSM4690537 SRP273093 PRJNA647773 SRS7076927 GSM4690537 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690537 GSM4690537 GEO Accession GSM4690537 SRX8814622 GSM4690538 SRP273093 PRJNA647773 SRS7076929 GSM4690538 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690538 GSM4690538 GEO Accession GSM4690538 SRX8814623 GSM4690539 SRP273093 PRJNA647773 SRS7076928 GSM4690539 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690539 GSM4690539 GEO Accession GSM4690539 SRX8814624 GSM4690540 SRP273093 PRJNA647773 SRS7076930 GSM4690540 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690540 GSM4690540 GEO Accession GSM4690540 SRX8814625 GSM4690541 SRP273093 PRJNA647773 SRS7076931 GSM4690541 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690541 GSM4690541 GEO Accession GSM4690541 SRX8814626 GSM4690542 SRP273093 PRJNA647773 SRS7076932 GSM4690542 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690542 GSM4690542 GEO Accession GSM4690542 SRX8814627 GSM4690543 SRP273093 PRJNA647773 SRS7076933 GSM4690543 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690543 GSM4690543 GEO Accession GSM4690543 SRX8814628 GSM4690544 SRP273093 PRJNA647773 SRS7076934 GSM4690544 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690544 GSM4690544 GEO Accession GSM4690544 SRX8814629 GSM4690545 SRP273093 PRJNA647773 SRS7076935 GSM4690545 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690545 GSM4690545 GEO Accession GSM4690545 SRX8814630 GSM4690546 SRP273093 PRJNA647773 SRS7076936 GSM4690546 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690546 GSM4690546 GEO Accession GSM4690546 SRX8814631 GSM4690547 SRP273093 PRJNA647773 SRS7076937 GSM4690547 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690547 GSM4690547 GEO Accession GSM4690547 SRX8814632 GSM4690548 SRP273093 PRJNA647773 SRS7076938 GSM4690548 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690548 GSM4690548 GEO Accession GSM4690548 SRX8814633 GSM4690549 SRP273093 PRJNA647773 SRS7076940 GSM4690549 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690549 GSM4690549 GEO Accession GSM4690549 SRX8814634 GSM4690550 SRP273093 PRJNA647773 SRS7076939 GSM4690550 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690550 GSM4690550 GEO Accession GSM4690550 SRX8814635 GSM4690551 SRP273093 PRJNA647773 SRS7076941 GSM4690551 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690551 GSM4690551 GEO Accession GSM4690551 SRX8814636 GSM4690552 SRP273093 PRJNA647773 SRS7076942 GSM4690552 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690552 GSM4690552 GEO Accession GSM4690552 SRX8814637 GSM4690553 SRP273093 PRJNA647773 SRS7076943 GSM4690553 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690553 GSM4690553 GEO Accession GSM4690553 SRX8814638 GSM4690554 SRP273093 PRJNA647773 SRS7076944 GSM4690554 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690554 GSM4690554 GEO Accession GSM4690554 SRX8814639 GSM4690555 SRP273093 PRJNA647773 SRS7076946 GSM4690555 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690555 GSM4690555 GEO Accession GSM4690555 SRX8814640 GSM4690556 SRP273093 PRJNA647773 SRS7076945 GSM4690556 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690556 GSM4690556 GEO Accession GSM4690556 SRX8814641 GSM4690557 SRP273093 PRJNA647773 SRS7076947 GSM4690557 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690557 GSM4690557 GEO Accession GSM4690557 SRX8814642 GSM4690558 SRP273093 PRJNA647773 SRS7076948 GSM4690558 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690558 GSM4690558 GEO Accession GSM4690558 SRX8814643 GSM4690559 SRP273093 PRJNA647773 SRS7076949 GSM4690559 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690559 GSM4690559 GEO Accession GSM4690559 SRX8814644 GSM4690560 SRP273093 PRJNA647773 SRS7076950 GSM4690560 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690560 GSM4690560 GEO Accession GSM4690560 SRX8814645 GSM4690561 SRP273093 PRJNA647773 SRS7076951 GSM4690561 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690561 GSM4690561 GEO Accession GSM4690561 SRX8814646 GSM4690562 SRP273093 PRJNA647773 SRS7076953 GSM4690562 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690562 GSM4690562 GEO Accession GSM4690562 SRX8814647 GSM4690563 SRP273093 PRJNA647773 SRS7076952 GSM4690563 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690563 GSM4690563 GEO Accession GSM4690563 SRX8814648 GSM4690564 SRP273093 PRJNA647773 SRS7076954 GSM4690564 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690564 GSM4690564 GEO Accession GSM4690564 SRX8814649 GSM4690565 SRP273093 PRJNA647773 SRS7076955 GSM4690565 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690565 GSM4690565 GEO Accession GSM4690565 SRX8814650 GSM4690566 SRP273093 PRJNA647773 SRS7076956 GSM4690566 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690566 GSM4690566 GEO Accession GSM4690566 SRX8814651 GSM4690567 SRP273093 PRJNA647773 SRS7076957 GSM4690567 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690567 GSM4690567 GEO Accession GSM4690567 SRX8814652 GSM4690568 SRP273093 PRJNA647773 SRS7076958 GSM4690568 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690568 GSM4690568 GEO Accession GSM4690568 SRX8814653 GSM4690569 SRP273093 PRJNA647773 SRS7076959 GSM4690569 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690569 GSM4690569 GEO Accession GSM4690569 SRX8814654 GSM4690570 SRP273093 PRJNA647773 SRS7076960 GSM4690570 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690570 GSM4690570 GEO Accession GSM4690570 SRX8814655 GSM4690571 SRP273093 PRJNA647773 SRS7076961 GSM4690571 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690571 GSM4690571 GEO Accession GSM4690571 SRX8814656 GSM4690572 SRP273093 PRJNA647773 SRS7076962 GSM4690572 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690572 GSM4690572 GEO Accession GSM4690572 SRX8814657 GSM4690573 SRP273093 PRJNA647773 SRS7076963 GSM4690573 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690573 GSM4690573 GEO Accession GSM4690573 SRX8814658 GSM4690574 SRP273093 PRJNA647773 SRS7076964 GSM4690574 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690574 GSM4690574 GEO Accession GSM4690574 SRX8814659 GSM4690575 SRP273093 PRJNA647773 SRS7076965 GSM4690575 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690575 GSM4690575 GEO Accession GSM4690575 SRX8814660 GSM4690576 SRP273093 PRJNA647773 SRS7076966 GSM4690576 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690576 GSM4690576 GEO Accession GSM4690576 SRX8814661 GSM4690577 SRP273093 PRJNA647773 SRS7076967 GSM4690577 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690577 GSM4690577 GEO Accession GSM4690577 SRX8814662 GSM4690578 SRP273093 PRJNA647773 SRS7076969 GSM4690578 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690578 GSM4690578 GEO Accession GSM4690578 SRX8814663 GSM4690579 SRP273093 PRJNA647773 SRS7076968 GSM4690579 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690579 GSM4690579 GEO Accession GSM4690579 SRX8814664 GSM4690580 SRP273093 PRJNA647773 SRS7076970 GSM4690580 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690580 GSM4690580 GEO Accession GSM4690580 SRX8814665 GSM4690581 SRP273093 PRJNA647773 SRS7076971 GSM4690581 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690581 GSM4690581 GEO Accession GSM4690581 SRX8814666 GSM4690582 SRP273093 PRJNA647773 SRS7076972 GSM4690582 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690582 GSM4690582 GEO Accession GSM4690582 SRX8814667 GSM4690583 SRP273093 PRJNA647773 SRS7076973 GSM4690583 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690583 GSM4690583 GEO Accession GSM4690583 SRX8814668 GSM4690584 SRP273093 PRJNA647773 SRS7076974 GSM4690584 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690584 GSM4690584 GEO Accession GSM4690584 SRX8814669 GSM4690585 SRP273093 PRJNA647773 SRS7076975 GSM4690585 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690585 GSM4690585 GEO Accession GSM4690585 SRX8814670 GSM4690586 SRP273093 PRJNA647773 SRS7076977 GSM4690586 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690586 GSM4690586 GEO Accession GSM4690586 SRX8814671 GSM4690587 SRP273093 PRJNA647773 SRS7076976 GSM4690587 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690587 GSM4690587 GEO Accession GSM4690587 SRX8814672 GSM4690588 SRP273093 PRJNA647773 SRS7076979 GSM4690588 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690588 GSM4690588 GEO Accession GSM4690588 SRX8814673 GSM4690589 SRP273093 PRJNA647773 SRS7076978 GSM4690589 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690589 GSM4690589 GEO Accession GSM4690589 SRX8814674 GSM4690590 SRP273093 PRJNA647773 SRS7076980 GSM4690590 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690590 GSM4690590 GEO Accession GSM4690590 SRX8814675 GSM4690591 SRP273093 PRJNA647773 SRS7076981 GSM4690591 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690591 GSM4690591 GEO Accession GSM4690591 SRX8814676 GSM4690592 SRP273093 PRJNA647773 SRS7076984 GSM4690592 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690592 GSM4690592 GEO Accession GSM4690592 SRX8814677 GSM4690593 SRP273093 PRJNA647773 SRS7076983 GSM4690593 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690593 GSM4690593 GEO Accession GSM4690593 SRX8814678 GSM4690594 SRP273093 PRJNA647773 SRS7076982 GSM4690594 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690594 GSM4690594 GEO Accession GSM4690594 SRX8814679 GSM4690595 SRP273093 PRJNA647773 SRS7076985 GSM4690595 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690595 GSM4690595 GEO Accession GSM4690595 SRX8814680 GSM4690596 SRP273093 PRJNA647773 SRS7076986 GSM4690596 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690596 GSM4690596 GEO Accession GSM4690596 SRX8814681 GSM4690597 SRP273093 PRJNA647773 SRS7076987 GSM4690597 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690597 GSM4690597 GEO Accession GSM4690597 SRX8814682 GSM4690598 SRP273093 PRJNA647773 SRS7076988 GSM4690598 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690598 GSM4690598 GEO Accession GSM4690598 SRX8814683 GSM4690599 SRP273093 PRJNA647773 SRS7076989 GSM4690599 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690599 GSM4690599 GEO Accession GSM4690599 SRX8814684 GSM4690600 SRP273093 PRJNA647773 SRS7076990 GSM4690600 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690600 GSM4690600 GEO Accession GSM4690600 SRX8814685 GSM4690601 SRP273093 PRJNA647773 SRS7076991 GSM4690601 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690601 GSM4690601 GEO Accession GSM4690601 SRX8814686 GSM4690602 SRP273093 PRJNA647773 SRS7076992 GSM4690602 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690602 GSM4690602 GEO Accession GSM4690602 SRX8814687 GSM4690603 SRP273093 PRJNA647773 SRS7076993 GSM4690603 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690603 GSM4690603 GEO Accession GSM4690603 SRX8814688 GSM4690604 SRP273093 PRJNA647773 SRS7076994 GSM4690604 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690604 GSM4690604 GEO Accession GSM4690604 SRX8814689 GSM4690605 SRP273093 PRJNA647773 SRS7076995 GSM4690605 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690605 GSM4690605 GEO Accession GSM4690605 SRX8814690 GSM4690606 SRP273093 PRJNA647773 SRS7076996 GSM4690606 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690606 GSM4690606 GEO Accession GSM4690606 SRX8814691 GSM4690607 SRP273093 PRJNA647773 SRS7076997 GSM4690607 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690607 GSM4690607 GEO Accession GSM4690607 SRX8814692 GSM4690608 SRP273093 PRJNA647773 SRS7076999 GSM4690608 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690608 GSM4690608 GEO Accession GSM4690608 SRX8814693 GSM4690609 SRP273093 PRJNA647773 SRS7076998 GSM4690609 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690609 GSM4690609 GEO Accession GSM4690609 SRX8814694 GSM4690610 SRP273093 PRJNA647773 SRS7077000 GSM4690610 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690610 GSM4690610 GEO Accession GSM4690610 SRX8814695 GSM4690611 SRP273093 PRJNA647773 SRS7077001 GSM4690611 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690611 GSM4690611 GEO Accession GSM4690611 SRX8814696 GSM4690612 SRP273093 PRJNA647773 SRS7077002 GSM4690612 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690612 GSM4690612 GEO Accession GSM4690612 SRX8814697 GSM4690613 SRP273093 PRJNA647773 SRS7077004 GSM4690613 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690613 GSM4690613 GEO Accession GSM4690613 SRX8814698 GSM4690614 SRP273093 PRJNA647773 SRS7077005 GSM4690614 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690614 GSM4690614 GEO Accession GSM4690614 SRX8814699 GSM4690615 SRP273093 PRJNA647773 SRS7077003 GSM4690615 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690615 GSM4690615 GEO Accession GSM4690615 SRX8814700 GSM4690616 SRP273093 PRJNA647773 SRS7077006 GSM4690616 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690616 GSM4690616 GEO Accession GSM4690616 SRX8814701 GSM4690617 SRP273093 PRJNA647773 SRS7077007 GSM4690617 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690617 GSM4690617 GEO Accession GSM4690617 SRX8814702 GSM4690618 SRP273093 PRJNA647773 SRS7077009 GSM4690618 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690618 GSM4690618 GEO Accession GSM4690618 SRX8814703 GSM4690619 SRP273093 PRJNA647773 SRS7077008 GSM4690619 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690619 GSM4690619 GEO Accession GSM4690619 SRX8814704 GSM4690620 SRP273093 PRJNA647773 SRS7077010 GSM4690620 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690620 GSM4690620 GEO Accession GSM4690620 SRX8814705 GSM4690621 SRP273093 PRJNA647773 SRS7077011 GSM4690621 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690621 GSM4690621 GEO Accession GSM4690621 SRX8814706 GSM4690622 SRP273093 PRJNA647773 SRS7077012 GSM4690622 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690622 GSM4690622 GEO Accession GSM4690622 SRX8814707 GSM4690623 SRP273093 PRJNA647773 SRS7077013 GSM4690623 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690623 GSM4690623 GEO Accession GSM4690623 SRX8814708 GSM4690624 SRP273093 PRJNA647773 SRS7077014 GSM4690624 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690624 GSM4690624 GEO Accession GSM4690624 SRX8814709 GSM4690625 SRP273093 PRJNA647773 SRS7077015 GSM4690625 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690625 GSM4690625 GEO Accession GSM4690625 SRX8814710 GSM4690626 SRP273093 PRJNA647773 SRS7077016 GSM4690626 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690626 GSM4690626 GEO Accession GSM4690626 SRX8814711 GSM4690627 SRP273093 PRJNA647773 SRS7077017 GSM4690627 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690627 GSM4690627 GEO Accession GSM4690627 SRX8814712 GSM4690628 SRP273093 PRJNA647773 SRS7077018 GSM4690628 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690628 GSM4690628 GEO Accession GSM4690628 SRX8814713 GSM4690629 SRP273093 PRJNA647773 SRS7077019 GSM4690629 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690629 GSM4690629 GEO Accession GSM4690629 SRX8814714 GSM4690630 SRP273093 PRJNA647773 SRS7077020 GSM4690630 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690630 GSM4690630 GEO Accession GSM4690630 SRX8814715 GSM4690631 SRP273093 PRJNA647773 SRS7077022 GSM4690631 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690631 GSM4690631 GEO Accession GSM4690631 SRX8814716 GSM4690632 SRP273093 PRJNA647773 SRS7077021 GSM4690632 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690632 GSM4690632 GEO Accession GSM4690632 SRX8814717 GSM4690633 SRP273093 PRJNA647773 SRS7077023 GSM4690633 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690633 GSM4690633 GEO Accession GSM4690633 SRX8814718 GSM4690634 SRP273093 PRJNA647773 SRS7077024 GSM4690634 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690634 GSM4690634 GEO Accession GSM4690634 SRX8814719 GSM4690635 SRP273093 PRJNA647773 SRS7077025 GSM4690635 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690635 GSM4690635 GEO Accession GSM4690635 SRX8814720 GSM4690636 SRP273093 PRJNA647773 SRS7077027 GSM4690636 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690636 GSM4690636 GEO Accession GSM4690636 SRX8814721 GSM4690637 SRP273093 PRJNA647773 SRS7077026 GSM4690637 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690637 GSM4690637 GEO Accession GSM4690637 SRX8814722 GSM4690638 SRP273093 PRJNA647773 SRS7077028 GSM4690638 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690638 GSM4690638 GEO Accession GSM4690638 SRX8814723 GSM4690639 SRP273093 PRJNA647773 SRS7077029 GSM4690639 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690639 GSM4690639 GEO Accession GSM4690639 SRX8814724 GSM4690640 SRP273093 PRJNA647773 SRS7077030 GSM4690640 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690640 GSM4690640 GEO Accession GSM4690640 SRX8814725 GSM4690641 SRP273093 PRJNA647773 SRS7077031 GSM4690641 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690641 GSM4690641 GEO Accession GSM4690641 SRX8814726 GSM4690642 SRP273093 PRJNA647773 SRS7077032 GSM4690642 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690642 GSM4690642 GEO Accession GSM4690642 SRX8814727 GSM4690643 SRP273093 PRJNA647773 SRS7077034 GSM4690643 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690643 GSM4690643 GEO Accession GSM4690643 SRX8814728 GSM4690644 SRP273093 PRJNA647773 SRS7077033 GSM4690644 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690644 GSM4690644 GEO Accession GSM4690644 SRX8814729 GSM4690645 SRP273093 PRJNA647773 SRS7077035 GSM4690645 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690645 GSM4690645 GEO Accession GSM4690645 SRX8814730 GSM4690646 SRP273093 PRJNA647773 SRS7077036 GSM4690646 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690646 GSM4690646 GEO Accession GSM4690646 SRX8814731 GSM4690647 SRP273093 PRJNA647773 SRS7077037 GSM4690647 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690647 GSM4690647 GEO Accession GSM4690647 SRX8814732 GSM4690648 SRP273093 PRJNA647773 SRS7077038 GSM4690648 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690648 GSM4690648 GEO Accession GSM4690648 SRX8814733 GSM4690649 SRP273093 PRJNA647773 SRS7077039 GSM4690649 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690649 GSM4690649 GEO Accession GSM4690649 SRX8814734 GSM4690650 SRP273093 PRJNA647773 SRS7077040 GSM4690650 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690650 GSM4690650 GEO Accession GSM4690650 SRX8814735 GSM4690651 SRP273093 PRJNA647773 SRS7077041 GSM4690651 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690651 GSM4690651 GEO Accession GSM4690651 SRX8814736 GSM4690652 SRP273093 PRJNA647773 SRS7077042 GSM4690652 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690652 GSM4690652 GEO Accession GSM4690652 SRX8814737 GSM4690653 SRP273093 PRJNA647773 SRS7077043 GSM4690653 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690653 GSM4690653 GEO Accession GSM4690653 SRX8814738 GSM4690654 SRP273093 PRJNA647773 SRS7077044 GSM4690654 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690654 GSM4690654 GEO Accession GSM4690654 SRX8814739 GSM4690655 SRP273093 PRJNA647773 SRS7077045 GSM4690655 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690655 GSM4690655 GEO Accession GSM4690655 SRX8814740 GSM4690656 SRP273093 PRJNA647773 SRS7077046 GSM4690656 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690656 GSM4690656 GEO Accession GSM4690656 SRX8814741 GSM4690657 SRP273093 PRJNA647773 SRS7077047 GSM4690657 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690657 GSM4690657 GEO Accession GSM4690657 SRX8814742 GSM4690658 SRP273093 PRJNA647773 SRS7077048 GSM4690658 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690658 GSM4690658 GEO Accession GSM4690658 SRX8814743 GSM4690659 SRP273093 PRJNA647773 SRS7077049 GSM4690659 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690659 GSM4690659 GEO Accession GSM4690659 SRX8814744 GSM4690660 SRP273093 PRJNA647773 SRS7077050 GSM4690660 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690660 GSM4690660 GEO Accession GSM4690660 SRX8814745 GSM4690661 SRP273093 PRJNA647773 SRS7077051 GSM4690661 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690661 GSM4690661 GEO Accession GSM4690661 SRX8814746 GSM4690662 SRP273093 PRJNA647773 SRS7077052 GSM4690662 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690662 GSM4690662 GEO Accession GSM4690662 SRX8814747 GSM4690663 SRP273093 PRJNA647773 SRS7077053 GSM4690663 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690663 GSM4690663 GEO Accession GSM4690663 SRX8814748 GSM4690664 SRP273093 PRJNA647773 SRS7077054 GSM4690664 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690664 GSM4690664 GEO Accession GSM4690664 SRX8814749 GSM4690665 SRP273093 PRJNA647773 SRS7077055 GSM4690665 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690665 GSM4690665 GEO Accession GSM4690665 SRX8814750 GSM4690666 SRP273093 PRJNA647773 SRS7077056 GSM4690666 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690666 GSM4690666 GEO Accession GSM4690666 SRX8814751 GSM4690667 SRP273093 PRJNA647773 SRS7077058 GSM4690667 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690667 GSM4690667 GEO Accession GSM4690667 SRX8814752 GSM4690668 SRP273093 PRJNA647773 SRS7077057 GSM4690668 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690668 GSM4690668 GEO Accession GSM4690668 SRX8814753 GSM4690669 SRP273093 PRJNA647773 SRS7077060 GSM4690669 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690669 GSM4690669 GEO Accession GSM4690669 SRX8814754 GSM4690670 SRP273093 PRJNA647773 SRS7077059 GSM4690670 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690670 GSM4690670 GEO Accession GSM4690670 SRX8814755 GSM4690671 SRP273093 PRJNA647773 SRS7077061 GSM4690671 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690671 GSM4690671 GEO Accession GSM4690671 SRX8814756 GSM4690672 SRP273093 PRJNA647773 SRS7077062 GSM4690672 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690672 GSM4690672 GEO Accession GSM4690672 SRX8814757 GSM4690673 SRP273093 PRJNA647773 SRS7077063 GSM4690673 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690673 GSM4690673 GEO Accession GSM4690673 SRX8814758 GSM4690674 SRP273093 PRJNA647773 SRS7077064 GSM4690674 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690674 GSM4690674 GEO Accession GSM4690674 SRX8814759 GSM4690675 SRP273093 PRJNA647773 SRS7077066 GSM4690675 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690675 GSM4690675 GEO Accession GSM4690675 SRX8814760 GSM4690676 SRP273093 PRJNA647773 SRS7077065 GSM4690676 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690676 GSM4690676 GEO Accession GSM4690676 SRX8814761 GSM4690677 SRP273093 PRJNA647773 SRS7077067 GSM4690677 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690677 GSM4690677 GEO Accession GSM4690677 SRX8814762 GSM4690678 SRP273093 PRJNA647773 SRS7077068 GSM4690678 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690678 GSM4690678 GEO Accession GSM4690678 SRX8814763 GSM4690679 SRP273093 PRJNA647773 SRS7077069 GSM4690679 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690679 GSM4690679 GEO Accession GSM4690679 SRX8814764 GSM4690680 SRP273093 PRJNA647773 SRS7077070 GSM4690680 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690680 GSM4690680 GEO Accession GSM4690680 SRX8814765 GSM4690681 SRP273093 PRJNA647773 SRS7077072 GSM4690681 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690681 GSM4690681 GEO Accession GSM4690681 SRX8814766 GSM4690682 SRP273093 PRJNA647773 SRS7077071 GSM4690682 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690682 GSM4690682 GEO Accession GSM4690682 SRX8814767 GSM4690683 SRP273093 PRJNA647773 SRS7077074 GSM4690683 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690683 GSM4690683 GEO Accession GSM4690683 SRX8814768 GSM4690684 SRP273093 PRJNA647773 SRS7077077 GSM4690684 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690684 GSM4690684 GEO Accession GSM4690684 SRX8814769 GSM4690685 SRP273093 PRJNA647773 SRS7077073 GSM4690685 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690685 GSM4690685 GEO Accession GSM4690685 SRX8814770 GSM4690686 SRP273093 PRJNA647773 SRS7077075 GSM4690686 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690686 GSM4690686 GEO Accession GSM4690686 SRX8814771 GSM4690687 SRP273093 PRJNA647773 SRS7077078 GSM4690687 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690687 GSM4690687 GEO Accession GSM4690687 SRX8814772 GSM4690688 SRP273093 PRJNA647773 SRS7077079 GSM4690688 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690688 GSM4690688 GEO Accession GSM4690688 SRX8814773 GSM4690689 SRP273093 PRJNA647773 SRS7077076 GSM4690689 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690689 GSM4690689 GEO Accession GSM4690689 SRX8814774 GSM4690690 SRP273093 PRJNA647773 SRS7077082 GSM4690690 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690690 GSM4690690 GEO Accession GSM4690690 SRX8814775 GSM4690691 SRP273093 PRJNA647773 SRS7077080 GSM4690691 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690691 GSM4690691 GEO Accession GSM4690691 SRX8814776 GSM4690692 SRP273093 PRJNA647773 SRS7077081 GSM4690692 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690692 GSM4690692 GEO Accession GSM4690692 SRX8814777 GSM4690693 SRP273093 PRJNA647773 SRS7077083 GSM4690693 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690693 GSM4690693 GEO Accession GSM4690693 SRX8814778 GSM4690694 SRP273093 PRJNA647773 SRS7077084 GSM4690694 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690694 GSM4690694 GEO Accession GSM4690694 SRX8814779 GSM4690695 SRP273093 PRJNA647773 SRS7077085 GSM4690695 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690695 GSM4690695 GEO Accession GSM4690695 SRX8814780 GSM4690696 SRP273093 PRJNA647773 SRS7077087 GSM4690696 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690696 GSM4690696 GEO Accession GSM4690696 SRX8814781 GSM4690697 SRP273093 PRJNA647773 SRS7077086 GSM4690697 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690697 GSM4690697 GEO Accession GSM4690697 SRX8814782 GSM4690698 SRP273093 PRJNA647773 SRS7077088 GSM4690698 RNA-Seq TRANSCRIPTOMIC cDNA Mice with LUAD tumors were euthanized at 2, 12, 20, or 30 weeks following tumor induction. We chose these time points because they reflect key stages in LUAD progression: atypical adenomatous hyperplasia (AAH) (KT and KPT at 2 weeks), adenoma (KT at 12 and 30 weeks), adenoma-to-LUAD transition (KPT at 12 weeks) and LUAD (KPT at 20 and 30 weeks). We micro-dissected large KPT tumors individually at 20 and 30 weeks, whereas all other samples were harvested by dissociating entire lungs containing mixtures of neoplasias in various stages of tumor progression. Following euthanasia, mice were perfused with S-MEM (Gibco, catalog #11380037) through the right ventricle of the heart. Dissected lungs or microdissected tumors were dissociated either with protease and DNAse solution in the Lung Dissociation Kit (Miltenyi Biotech, catalog #130-095-927) followed by mechanical dissociation using gentleMACS “C” columns (Miltenyi Biotech, catalog #130-093-237) according to the manufacturer's instructions (Tammela et al., 2017), or by a mixture of Dispase II (Gibco, catalog #17105-041, final concentration 0.6 U/ml), Collagenase Type IV (Thermo Fisher Scientific, catalog #17104019; final concentration 0.083 U/ml), and DNase I (Sigma-Aldrich, catalog #69182-3; final concentration 10 U/ml) in S-MEM solution containing Gentamicin (Gibco, catalog #15750-060, final concentration 20 mg/ml) at 37°C for 30 min (Table S6). The dissociated cells were filtered using a 100 mm strainer and spun at 300 g for 5 min at room temperature. The supernatant was removed by aspiration and red blood cell lysis was performed using ACK (Thermo Fisher Scientific, catalog #A1049201). Cells were then washed with media and pelleted at 300 g for 5 min at 4°C. The supernatant was removed, and the pellet resuspended in Fluorescence-Activated Cell Sorting (FACS) buffer media (200 mM EDTA with 250 ml heat-inactivated FBS in PBS) before being passed through a 40 mm strainer and counted for use in FACS below. Cells were dissociated as above, stained with DAPI and live cells were sorted as described above into 96 well plates containing 5 ml of TCL Buffer (Qiagen, catalog #1031576) with 1% beta-mercaptoethanol. Plates were processed by a modified SMART-Seq2 protocol (Picelli et al., 2013), with the following modifications: RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter, catalog #A63881) using Bravo Automated Liquid Handling Platform prior to oligo-dT primed reverse transcription with Maxima reverse transcriptase (Thermo Fischer, catalog #EP0752) and locked TSO oligonucleotide (Exiqon, custom made), which was followed by a 21 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, catalog #KK2601). The WTA product was purified using Agencourt AMPure XP beads (Beckman-Coulter, catalog #A63881) and a Bravo Automated Liquid Handling Platform. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina, catalog #FC-131-1096) with custom barcode adapters. Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina, catalog #FC-404-2005) at the Broad Genomics Platform. SmartSeq2 NextSeq 500 gds 304690698 GSM4690698 GEO Accession GSM4690698